\=>' 


LIBRARY 

OF    THE 

UNIVERSITY  OF  CALIFORNIA, 


ClaSS  LIBRARY 


•»  -.   . 


r* 


A   LABORATORY  GUIDE   IN 
BACTERIOLOGY 


A  Laboratory  Guide 
in  Bacteriology 


FOR  THE  USE  OF 


STUDENTS,  TEACHERS,  AND 
PRACTITIONERS 


BY 


PAUL  G.  HEINEMANN,  Sc.B. 

Fellow  in  Bacteriology,  the  University  of  Chicago 


THE  UNIVERSITY  OF  CHICAGO  PRESS 
1905 


t+4- 


COPYRIGHT   1905 
THE  UNIVERSITY  OF  CHICAGO 


PREFACE 

The  considerations  which  led  the  writer  to  add  this 
laboratory  guide  of  bacteriology  to  the  number  of  such 
guides  already  in  existence  were  of  various  nature  and 
may  be  briefly  set  forth  here. 

Probably  no  branch  of  biological  science  has  advanced 
so  rapidly  during  the  past  few  years  as  the  science  of 
bacteriology,  and  it  is  difficult  even  for  an  active  labora- 
tory worker  to  keep  abreast  of  this  advance.  A  text- 
book or  guide  fixes  the  status  of  the  science  at  the  time 
of  its  writing,  but  almost  before  it  leaves  the  press  it 
becomes  antiquated.  Revisions,  corrections,  and  addi- 
tions are  necessary  at  short  intervals  in  order  to  keep  a 
publication  of  this  kind  approximately  up  to  date.  There 
is  therefore,  almost  at  any  given  moment  room  for  a  new 
publication  to  fill  the  want  of  a  progressive  instructor  for 
a  guide  that  gives  the  latest  accepted  rules  and  practices 
of  the  laboratory.  The  value  of  such  a  publication  will 
be  enhanced  by  a  plan  and  arrangement  of  sufficient 
flexibility  and  latitude  to  allow  the  instructor  and  the 
student  to  enter  such  additions  and  corrections  as  serve 
to  bridge  over  the  time  between  editions. 

Medical  students  entering  on  a  course  in  bacteriology 
often  have  had  too  little  previous  laboratory  training  in 
methods  of  precision.  It  is  a  matter  of  importance  for 
the  instructor  to  put  himself  in  the  attitude  of  mind  of  the 
student  and  to  try  to  appreciate  his  difficulties  in  under- 
standing details.  Many  of  the  pieces  of  apparatus 
employed  in  a  bacteriological  laboratory  are  novel  even 


144184 


vi  PREFACE 

to  the  student  trained  in  chemistry  and  biology,  and  it 
has  been  thought  best  to  exhibit  these,  to  the  smallest 
detail,  by  means  of  illustrations — a  feature  not  sufficiently 
considered  in  other  guides. 

The  formulae  for  stains  and  the  methods  of  staining 
have  not  been  collected  in  one  chapter,  as  is  usually  the 
practice,  because  this  tends  to  confuse  the  student. 
They  are  described  during  the  progress  of  the  course,  as 
occasion  offers  to  put  them  to  practical  use. 

Culture-description  charts  have  not  been  included  in 
this  volume.  A  beginner  naturally  makes  incomplete 
descriptions  and  many  alterations,  and  thus  defaces  the 
book  and  impairs  its  future  utility.  A  sufficient  number 
of  loose  charts  perforated  for  binding  should  be  furnished 
to  the  student  at  a  nominal  figure. 

A  point  of  inestimable  importance  is  how  best  to 
stimulate  the  student  to  consult  textbooks  and  special 
monographs,  and  other  references,  as  often  and  as  freely 
as  possible.  This  guide  has  been  written  with  the  aim 
of  not  only  not  interfering  in  any  manner  with  the  reading, 
through  including  such  points  and  characteristics  as  might 
make  a  textbook  superfluous  in  the  judgment  of  the 
inexperienced,  but  also  of  making  it  necessary  for  the 
student  to  read  the  best  textbooks  with  freedom  and 
understanding.  Cultural  and  morphological  features  are 
left  entirely  to  the  actual  observation  of  the  student,  sup- 
plemented by  instruction  and  the  reading  of  textbooks. 

The  course,  as  outlined,  is  identical  with  the  medical 
course  given  at  the  University  of  Chicago,  with  a  few 
additional  chapters  which  may  be  used  during  courses 
for  non-medical  students.  A  chapter  containing  a  fairly 
complete  list  of  formulae  for  culture  media  employed  in 


PREFACE  vii 

advanced  work  has  been  added  with  the  object  of  making 
them  easily  accessible  to  those  engaged  in  advanced  work. 
The  laboratory  guides  of  Novy,  Eyre,  Frost,  Gorham, 
Kanthack  and  Drysdale,  and  Connell,  and  the  American 
edition  of  the  Manual  oj  Bacteriology  of  Muir  and  Ritchie, 
have  been  freely  consulted.  I  take  this  occasion  of 
expressing  my  deep  gratitude  to  Professor  Edwin  O. 
Jordan  and  Dr.  Norman  Mac  Leod  Harris  for  their 
invaluable  help  and  suggestions  in  the  preparation  of 
this  guide. 

PAUL  G.  HEINEMANN. 
CHICAGO,  ILL. 
June,  1905. 


INTRODUCTION 

The  advent  of  bacteriology  into  the  realm  of  the 
biological  sciences  not  only  brought  with  it  a  new 
conception  of  the  nature  of  many  complicated  phe- 
nomena, such  as  fermentation  and  disease,  but  also 
placed  in  the  hands  of  experimental  workers  a  new 
tool.  The  method  of  sterilization,  of  asepsis,  made 
it  possible  for  the  first  time  to  attack  problems 
hitherto  incapable  of  solution,  or  even  of  approach. 
This  development  of  bacteriological  technique,  of 
rigid  and  undeviating  adherence  to  definite  rules 
and  principles,  is  not  likely  to  be  passed  over  lightly 
by  the  historian  of  nineteenth-century  science.  The 
art  of  practical  medicine  and  theoretical  medical 
research  alike  owe  much  of  their  recent  brilliant 
success  to  a  ready  adoption  of  the  new  method. 

At  the  present  time  an  active  campaign  is  being 
set  on  foot  by  public  health  authorities  against 
several  widespread  and  serious  diseases  of  the  human 
race.  In  various  parts  of  the  world,  malaria,  tuber- 
culosis, and  typhoid  fever  are  being  fought  energeti- 
cally and  with  much  success.  In  these  systematic 
and  organized  movements  the  resources  of  bacteri- 
ology are  being  utilized  as  never  before,  and  a  full 
understanding  of  technical  procedure  and  devices 
is  deemed  essential  by  all  workers  in  this  subject. 
The  problems  of  water-supply  and  sewage  disposal, 

ix 


x  INTRODUCTION 

of  urban  infantile  mortality,  and  of  the  control  of 
contagious  diseases  are  all  bound  up  with  the  intelli- 
gent application  of  bacteriological  methods. 

In  the  almost  untilled  field  of  industrial  bacteri- 
ology there  is  need  for  a  fuller  appreciation  of  the 
value  of  bacteriological  methods  and  principles. 
Many  great  industries  are  based  wholly  upon  the 
proper  selection  and  adaptation  of  micro-organisms, 
and  a  timely  and  discriminating  utilization  of  their 
products.  Loose  and  empirical  methods  have  been 
in  force  in  the  past,  but  these  must  eventually  give 
way  to  a  more  precise  and  truly  bacteriological 
technique. 

Agricultural  bacteriology  is  just  now  much  in  the 
public  eye,  and  it  would  be  gratuitous  to  prophesy 
the  results  that  may  reasonably  be  anticipated  in 
this  direction.  Here  again  crude,  rule-of-thumb, 
" practical"  ways  of  doing  things  are  being  sup- 
planted by  the  scientific,  the  reasoning,  and  the 
precise. 

To  the  student,  whether  in  medical,  hygienic,  or 
industrial  bacteriology,  proper  technical  methods 
of  work  must  always  have  a  peculiar  value,  since 
without  their  aid  advance  is  impossible,  and  stumb- 
ling and  disastrous  missteps  are  certain.  A  com- 
prehensive outline  of  modern  bacteriological  methods, 
therefore,  is  a  necessary  adjunct  to  obtaining  a  true 
and  full  understanding  of  the  underlying  principles 
and  tendencies  of  the  science.  The  technique  of 


INTRODUCTION  xi 

bacteriology  is  one  of  its  greatest  contributions  to 
both  science  and  art,  and  the  use  of  so  valuable 
and  simple  a  tool  should  be  mastered  not  only  by 
the  biological  teacher  and  investigator,  but  by  prac- 
tical workers  in  medicine,  hygiene,  and  many  other 
fields. 

EDWIN  O.  JORDAN. 


TABLE  OF  CONTENTS 

PAGE 

CHAPTER  I.    Laboratory  Rules     .         .         .         .         .  .       i 

CHAPTER  II.    Cleaning,  Preparing,  and  Sterilizing  Glassware      8 

CHAPTER  III.   Preparation  of  Culture  Media     .          .  .11 

Exercise  i.     Preparation  of  nutrient  agar-agar      .  .11 

Exercise  2.     Preparation  of  glucose-agar      .          .  -19 

Exercise  3.     Preparation  of  peptone-gelatin           .  -23 

Exercise  4.     Preparation  of  peptone-broth             .  -25 

Exercise  5.     Preparation  of  potato       .          .          .  .27 

Exercise  6.     Preparation  of  litmus  milk        .          .  .28 

CHAPTER  IV.    Preparation  of  Staining  Solutions          .  .     30 

CHAPTER  V.   The  Microscope            .          .          .          .  -31 

CHAPTER  VI.    Collecting  Bacteria  from  the  Air  .          .  -37 

CHAPTER  VII.    Exercises  on  Infection  and  Sterilization  .     45 

Exercise  i.     Phenomena  of  infection             .          .  -45 

Exercise  2.     Phenomena  of  sterilization        .          .  -45 

Exercise  3.     Phenomena  of  sterilization  (continued)  .     45 

CHAPTER  VIII.    Study  of  Yeasts,  Molds,  and  Torulae  .     49 

Exercise  i.     Cultural  studies       .          .          .          .  -49 

Exercise  2.     Study  of  the  germination  of  spores    .  51 

CHAPTER  IX.   Scheme  for  Routine   Study  of   the  Various 

Groups  of  Organisms         .          .          ,          .          .  -53 

CHAPTER  X.    Method  of  Describing  Cultures     .          .  -57 

CHAPTER  XI.    Study  of  Certain  Chromogenic  Bacteria  .     69 

Exercise  i.     Cultural  studies      .  '        .          .          .  .69 

Exercise  2.     Study  of  pigments  .                   .          .  .     71 

CHAPTER  XII.   The  Pyogenic  Group         .         .  -73 

Exercise  i.     Subgroup  A             .         .         .         .  -73 
Exercise  2.     Subgroup  B             .         ....     76 


xiv  TABLE  OF  CONTENTS 

PAGE 

CHAPTER  XIII.  The  Intestinal  Group       .          .          .          -79 

Exercise  i.  Colon  group  .          .          .          .          .          -79 

Exercise  2.  Hog-cholera,  Bac.  enteritidis  or  intermediate 

group  .                                                                         .      84 

Exercise  3.  Typhoid-dysentery  group          .                         85 

Exercise  4.  Proteus  group          .          .          .          .          .89 

CHAPTER  XIV.   The  Capsulated  Group    .          .          .  .92 

CHAPTER  XV.    The  Diphtheria  Group      .         .          .  -94 

CHAPTER  XVI.    The  Hemorrhagic  Septicemia  Group.  .     97 

CHAPTER  XVII.   The  Anthrax  Group        .          .  .98 

CHAPTER  XVIII.   The  Spirillum  Group    .          .          .  .    101 

CHAPTER  XIX.   The  Group  of  Acid-Resisting  Bacilli  .    103 

CHAPTER  XX.   The  Miscellaneous  Bacteria        .          .  .    105 

CHAPTER  XXI.   The  Actinomyces  Group  .          .  .    106 

CHAPTER  XXII.   The  Anaerobic  Group  of  Bacilli       .  .    107 

CHAPTER  XXIII.    Isolation  of  Unknown  Bacteria    from   a 

Mixture  .          .          .          .          .          .          .          .          .112 

CHAPTER  XXIV.    Bacteriological  Examination  of  Water,  Air, 

and  Milk  .          .          .          .          .          .          .          .114 

Exercise  i.     Bacteriological  examination  of  water  .    114 

Exercise  2.  Bacteriological  examination  of  air  .  .118 
Exercise  3.  Bacteriological  examination  of  milk  .  119 

CHAPTER  XXV.    Influence  of  Disinfectants  on  the  Growth 

of  Micro-organisms 122 

Exercises  i  and  2.  Hill's  test-rods  .  .-»-,.  .  .122 
Exercise  3.  Influence  ot  sunlight  .  *.^?^-  •  •  I23 
Exercise  4.  Influence  of  moist  heat  .  .  .  .124 

APPENDIX  I.    Special  Media 127 

APPENDIX  II.    Staining  Solutions 132 

APPENDIX  III.    Frost's  Culture  Chart  (modified)       >         .134 
INDEX 139 


CHAPTER  I 
LABORATORY  RULES 

1.  Carefully  familiarize  yourself  with  the  laboratory 
rules.     Upon  their  careful  observance  depend  good  work 
and  your  own  safety. 

2.  Food  must  not  be  eaten  in  the  laboratory;  lead- 
pencils,  labels,  or  fingers  must  not  be  moistened  with 
the  tongue. 

3.  If  any  portion  of  a  culture  is  spilled  by  accident 
upon  the  desk  or  floor,  it  should  be  immediately  covered 
with  a  germicide  (HgCl2  i :  1000  or  5  per  cent,  carbolic 
acid).     After  this  has  acted  for  10  or  15  minutes,  wipe  it 
up  and  throw  the  cloth  or  paper  into  a  waste-jar. 

4.  In  case  the  hands  should  by  accident  come  in 
contact  with  infectious  material,  they  should  be  washed 
with  one  of  the  above-mentioned  germicides,  and  then 
thoroughly  scrubbed  with  soap  and  water. 

5.  The  platinum  needles  used  in  making  cultures 
should  be  sterilized  in  a  flame  shortly  before  and  imme- 
diately after  use,  and  before  they  are  laid  down.     When 
the  needles  are  covered  with  moist  infectious  material, 
they  should  be  held  at  the  side  of  the  flame  until  dry 
before  being  sterilized;    this  will  avoid  the  danger  of 
scattering  this  material  about  the  desk. 

6.  All  possible  care  should  be  observed  in  the  care 
of  the  apparatus,  desk,  etc.     Solid  materials  must  not 
be  put  into  the  sinks.     Burned  matches,  paper,  cotton, 
etc.,  are  to  be  put  in  the  crocks  provided  for  that  purpose, 
and  not  on  the  floor. 


2         LABORATORY   GUIDE   IN   BACTERIOLOGY 

7.  Discarded  cultures  should  be  killed  in  the  auto- 
clav  (5  minutes  at  I2o0j)  before  emptying  into  the  crocks. 

8.  When  using  the  steam  sterilizers  and   autoclav, 
see  that  the  pan  contains  sufficient  water  before  lighting 
the  gas. 

9.  In  general,  see  that  the  air  inlets  of  Bunsen  burners 
are  open  before  lighting,  and  relight  if  the  flame  strikes 
down. 

10.  Always  return  stock-bottles  to  the  proper  places 
on  the  shelves.    • 

11.  At  the  close  of  the  day's  work  the  desks  should 
be  washed  off  with  corrosive  sublimate,  and  the  hands 
cleaned  by  thorough  scrubbing  with  green   soap  and 
water. 

12.  Before  leaving  the  laboratory,  see  that  the  gas 
is  shut  off  under  all  apparatus,  that  water  faucets  are 
closed,  and  that  all  glassware,  etc.,  is  replaced  in  the 
lockers. 

GENERAL  DIRECTIONS 

1.  After  obtaining  the  key  to  the  locker,  examine  the 
outfit,  check  the  apparatus  (Fig.  i)  on  the  furnished  list, 
and  return  the  same  signed  with  your  name.     This  signa- 
ture is  a  receipt  in  full  for  the  material  received  and  in 
good  condition,  and  the  student  is  personally  responsible 
for  the  return  of  the  same  in  good  condition. 

2.  Matches,   peptone,  gelatin,  and   filter-paper  are 
furnished  with  the  locker  outfit  in  sufficient  quantities 
to  last  through  the  course.     Anything  needed  in  excess 
must  be  furnished  by  the  student. 

1  Degrees  of  temperature  throughout  this  work  are  indicated 
according  to  the  Centigrade  system. 


LABORATORY  RULES  3 

3.  To  facilitate  study,  students  may  work  in  pairs  in 
the  preparation  of  culture  media  and  the  cultivation  of 
organisms;   but  each  student  is  responsible  for  a  first- 
hand  and   independent   description   of  each   organism 
studied,  as  well  as  for  stained  cover-slip  preparations. 

4.  Read  carefully  in  the  textbook  and  the  laboratory 
guide  the  work  for  the  day.     This  will  greatly  facilitate 
an  intelligent  and  systematic  plan  of  work. 

5.  Keep  careful,  neat,  systematic  notes  of  each  exer- 
cise, and  have  them  ready  for  inspection  at  any  time. 
In  the  case  of  exercises  devoted  to  the  preparation  of 
culture  media,  a  simple  statement  as  to  their  completion 
is  sufficient. 

6.  Printed  charts  for  description  of  cultures  can  be 
obtained  on  application  to  the  laboratory  assistant. 

7.  Accurate  sketches  must  be  made  when  called  for 
by  the  directions. 

8.  All  the  notes  pertaining  to  each  exercise  must  be 
kept  together.     "University  covers"  of  suitable  size  are 
recommended  for  this  purpose. 

9.  Store  clean  cover-slips  in  a  Stender  dish  and  cover 
with  96  per  cent,  alcohol.     A  soft  linen  cloth  is  necessary 
for  wiping  them.     An  ordinary  clean  handkerchief  will 
do  very  well.     Coarse  material  breaks  them  easily. 

LOCKER  OUTFIT 

150  culture-tubes. 
10  potato  culture-tubes. 
6  fermentation-tubes. 
15  Petri  dishes  (for  plate-cultures). 

3  Erlenmeyer  flasks,  one  1000  c.c.,  two  500  c.c.  each. 
2  glass  funnels,  one  4  inches,  one  6  inches. 

4  bottles  for  staining-fluiols, 


LABORATORY   GUIDE   IN   BACTERIOLOGY 


Retort-Stand 


Bunsen  Burner 
FiO.  i 


Culture-Tube          Pipette 


LABORATORY  RULES 


Petri  Dish 


Tripod 


Watch-G  lasses 


Erlenmeyer  Flask 


Saltcellar 


Balsam  Bottl 


Magnifier 
(Hand-Lens) 

FIG.  i 


Slender  Dish 


6        LABORATORY  GUIDE   IN   BACTERIOLOGY 

i  balsam  bottle. 

i  Stender  dish  (for  cover-slips). 

3  watch-glasses, 
i  saltcellar. 

1  glass  rod. 

2  platinum  needles  (turn  the  end  of  one  needle  around 
a  sharp  pencil  point  so  as  to  form  a  closed  loop). 

8  tin  cups  or  tumblers  (cover  the  bottom  of  each  with 
ordinary  cotton). 

4  wire  baskets  (to  hold  culture-tubes). 
i  Bunsen  burner  and  rubber  tubing. 

i  saucepan  and  cover. 

3  graduates,  one  500  c.c.,  one  100  c.c.,  one  10  c.c. 
i  pinchcock. 

i  pipette  with  rubber  hose  attached, 
i  magnifier  (hand-lens), 
i  tripod. 

i  retort-stand  with  three  rings, 
i  thermometer  in  case, 
i  box  of  matches. 
40  grams  of  peptone. 
1 20  grams  of  gelatin. 
6  sheets  of  filter-paper, 
i  key. 

This  outfit  may  be  changed  and  added  to  in  the  dis- 
cretion of  the  instructor. 

Obtain  the  following  articles  from  the  storeroom: 
50  glass  slides. 

1  camel's-hair  brush. 

50  round  cover-glasses  (18  mm.). 

2  towels  (one  should  be  boiled  in  dilute  NaOH,  rinsed 
in  clean  water,  and  kept  clean  for  wiping  slides  and 

cover-glasses.) 


LABORATORY  RULES 

1  test-tube  brush. 
50  labels. 

2  slide-boxes. 

2  hollow-ground  slides. 

i  glass  pencil. 

i  pair  of  forceps. 

i,  or  better  2,  pairs  of  cover-slip  forceps. 


CHAPTER  II 

CLEANING,  PREPARING,  AND  STERILIZING 
GLASSWARE 

EXERCISE  I.      CLEANING  GLASSWARE 

Culture-tubes,  flasks,  fermentation-tubes,  and  Petri 
dishes  must  be  free  from  organic  matter,  acids,  and 
alkalis.  They  are  best  cleaned  as  follows: 

a)  Completely  immerse  them  in  a  vessel  containing 
soapsuds  or  soap-powder,  boil  for  at  least  10  minutes, 
then  thoroughly  brush  them  with  the  tube-brush;    or, 
immerse  them  for  an  hour  or  more  in  the  chromic-acid 
cleaning-mixture,  consisting  of— 

Potassium  dichromate 60  parts 

Water 300  parts 

Concentrated  sulphuric  acid 460  parts 

The  sulphuric  acid  is  to  be  added  slowly  with  constant 
stirring. 

b)  Rinse  thoroughly  in  tap-water. 

c)  Again  use  the  tube-brush,  and  soap  and  water,  if 
necessary. 

d)  Rinse  very  thoroughly,  and  get  rid  of  every  trace 
of  acid,  soap,  or  alkali. 

e)  Turn  upside  down  in  a  basket,  and  heat  for  20 
minutes  in  the  hot-air  sterilizer  until  perfectly  dry. 

EXERCISE  II.      PLUGGING  OF  CULTURE-TUBES 

Directions  for  plugging. — Take  a  small  amount  of 
ordinary,  non-absorbent  cotton  of  good  quality,  fold  it 
once,  and  by  means  of  a  glass  rod  push  it  into  the  mouth 

8 


CLEANING   AND   PREPARING   GLASSWARE 


of  the  tube  to  the  depth  of  about  J  of  an  inch;    or  roll 
the  cotton  into  cylinders  of  the  same      s^*^/^ 
diameter  as  the  culure-tube  and  insert    /  ^      ~ 
it  into  the  mouth  (Fig.   2).    The  plug  /f7r  ^ 
should  allow  the  interchange  of  air, 
and  at  the  same  time  be  tight  enough 
to  hold  the  weight  of  the  tube,  and 
with  no  crevices  on  the  side.     The 
glass    rod   should   never  be    pushed 
through  the  cotton. 

EXERCISE  III  Flo.  3 

STERILIZATION  OF  GLASSWARE  Plugged  Culture-Tube 

Sterilization  is  the  process  of  killing  all  micro-organ- 
isms. This  may  be  accomplished  by  heat,  by  certain 
chemicals,  or  by  nitration.  Sterilization  by  heat  may  be 
accomplished — 

1.  By  dry  heat;  this  method  is  applied  to  sterilization 
of  many  kinds  of  apparatus. 

2.  By  moist  heat;   this  method  is  applied  largely  to 

sterilization  of  culture  media, 
and  will  be  described  in 
Chap.  Ill,  Exercise  II. 

Hot-air  sterilizers  are  boxes 
with  double  walls  of  sheet- 
iron.  The  bottom  shelf 
should  always  be  covered  with 
a  piece  of  sheet-asbestos,  to 
prevent  too  rapid  heating  of 
the  apparatus.  By  means  of 
a  Bunsen  burner  with  three 
flames  a  heat  of  about  150° 
may  easily  be  maintained.  The  flames  of  the  burner 


FIG.  3 
Koch's  Hot-Air  Sterilizer 


io       LABORATORY   GUIDE   IN   BACTERIOLOGY 

should  enter  the  hole  provided  at  the  bottom  of  the 
sterilizer  for  that  purpose.  Care  should  be  taken  to 
avoid  the  possibility  of  the  flame  becoming  luminous, 
as  this  would  fill  the  box  and  cover  the  glassware  with 
soot  (Figs.  3  and  4). 

The  plugged  culture-tubes  are  then  placed  in  the 


FIG.  4 
Lautenschlager  Hot-Air  Sterilizer 

sterilizer,  and  a  temperature  of  150°  is  maintained  for 
about  30  minutes,  or  until  the  plugs  are  slightly  charred. 
This  can  be  recognized  by  a  slight  brownish  color.  The 
tubes  are  now  not  necessarily  sterile,  but  the  plugs  have 
become  set  so  as  to  fit  the  mouth  of  the  tube,  and  may 
easily  be  removed  and  replaced. 


CHAPTER  III 

PREPARATION  OF  CULTURE  MEDIA 
Each  student  is  required  to  make  up  the  following 
amounts  of  culture  media: 


Name  of  Medium 

Amount 

Number  of  Tubes 

Plain  agar.  ... 

IOOO  2 

•7Q 

G  lucose-agar  . 

2CO  £ 

20 

Gelatin.  . 

JQQ   or  . 

2  ^ 

Potato  

a****  &• 

*0 

IO 

Broth  

300  c.c. 

2C 

Dunham's  solution. 

?OO    C   C. 

2cr 

Litmus  milk. 

7OO  C  C 

2^ 

*3 

160 

EXERCISE    I.      PREPARATION     OF     NUTRIENT     AGAR-AGAR 

1.  Weigh  the  saucepan  without   the  lid  accurately 
and  make  note  of  the  weight. 

2.  Measure  into  the  saucepan  1000  c.c.  of  tap- water, 
adding  about  300  c.c.  of  water  to  allow  for  evaporation, 
and  heat  over  gas. 

3.  Cut  and  shred  i<;g.  of  agar-agar  (ij  per  cent.), 
add  to  the  water,  and  boil  slowly,  with  constant  stirring, 
until  perfectly  dissolved,  taking  care  not  to  overheat. 

4.  Add  3  g.   of   extract  of    beef.     When  dissolved, 
remove  the  saucepan  from  the  flame,  and 

5.  Slowly  dust  10  ( i  per  cent.)  g.  of   Witte's  peptone 
on  the  surface,  constantly  stirring  until  perfectly  dissolved. 
Avoid  allowing  the  peptone  to  clump. 

NOTE. — Agar-agar  (called  simply   "agar")  is   a  watery  ex- 
tract of  certain  seaweeds  found  on  the  Pacific  coast  of  Asia. 


12       LABORATORY   GUIDE   IN   BACTERIOLOGY 

6.   Adjust  the  reaction. 

Bacteria,  especially  pathogenic  bacteria,  grow  prefer- 
ably in  a  medium  which  is  neutral  or  slightly  acid  to 
phenolphthalein  (alkaline  to  litmus).  Extract  of  beef 
always  contains  a  certain  amount  of  acid,  which  has  to 
be  neutralized.  Two  methods  may  be  employed  to 
accomplish  this  purpose. 

First  method. — Gradually  add  a  4  per  cent,  solution 
of  sodium  hydrate  (NaOH)  until  a  sample  on  the  end 
of  the  glass  rod  turns  phenolphthalein  paper  a  pale  rose- 
pink  color.  If  accidentally  too  much  NaOH  is  added, 
it  may  be  readjusted  by  means  of  a  5  per  cent,  solution 
of  hydrochloric  acid  (HC1).  This  method  is  sufficiently 
accurate  for  ordinary  purposes.  The  medium  is  then 
strongly  alkaline  to  litmus. 

Second  method. — A  more  precise  method  is  as  follows: 
By  means  of  a  pipette  measure  5  c.c.  of  the  liquid  into 
a  porcelain  evaporating-dish ;  add  45  c.c.  of  distilled 
water  and  i  c.c.  of  a  solution  of  i  part  phenolphthalein 
in  200  c.c.  alcohol  (50  per  cent.),  then  heat  to  the  boiling- 
point  and  slowly  add  from  a  graduated  burette  enough 
-£$  normal  NaOH  to  neutralize.  (The  liquid  must 
have  a  decided,  stable,  pale-pink  color  which  does  not 
vanish  when  heated.)  Read  the  amount  used  from  the 
burette,  and  calculate  the  amount  of  normal  NaOH  needed 
for  1000  c.c.  Then  add  0.5  per  cent,  normal  HC1. 

Example. — By  reading  the  burette  we  find  that  it 
takes  1.2  c.c.  -£$  normal  NaOH  to  neutralize  5  c.c.  of 
the  medium,  diluted  with  45  c.c.  of  distilled  water.  To 
neutralize  i  liter,  it  will  take  200  times  this  amount, 
which  amounts  to  240  c.c.  of  the  -^  normal  NaOH. 
Divide  240  by  20,  and  the  result  (12  c.c.)  is  the  amount 


PREPARATION   OF    CULTURE   MEDIA  13 

of  normal  NaOH  needed  to  neutralize  i  liter  of  medium. 
To  this  add  5  c.c.  normal  HCL 

The  medium  is  then  0.5  per  cent,  acid  to  phenol- 
phthalein  (  +  0.5  per  cent.  Fuller's  standard),  or  fairly 
strongly  alkaline  to  litmus. 

The  same  result  may  be  obtained  by  deducting  5  c.c. 
from  the  amount  of  normal  NaOH  to  be  added  per  liter. 
If,  therefore,  in  the  above  example  12  c.c. —  5  c.c.  =  7  c.c. 
normal  NaOH  is  added,  the  resulting  reaction  of  the  me- 
dium is  0.5  per  cent,  acid  without  the  addition  of  HCL 

A  normal  solution  is  the  equivalent  weight  in  grams 
(gram-molecule)  of  a  chemical  in  1000  c.c.  of  distilled 
water.  In  the  case  of  monovalent  elements  combining 
to  form  the  chemical,  the  molecular  weight  of  the  latter  is 
taken;  in  the  case  of  bivalent  ones,  the  molecular 
weight  is  divided  by  2 ;  etc. 

7.  Allow  the  liquid  to  cool  to  60°;   test  by  placing 
the  thermometer  in  it. 

8.  While  cooling,  dissolve  the  whites  of  two  eggs  in 
75  c.c.  of  water,  and  stir  well  into  the  liquid  while  still  at 
60°.    The  egg  albumen  is  added  for  the  purpose  of  clari- 
fying the  liquid.     If  added  before  the  temperature  has 
fallen  to  60°,  it  would  partially  coagulate,  and  thus  not 
serve  the  purpose.     On  the  other  hand,  if  allowed  to 
cool  to  about  40°,  the  agar  would  solidify.     Upon  gradu- 
ally heating  the  medium  the  albumen  coagulates,  me- 
chanically incloses  suspended  particles,  carries  them  to 
the  top,  and  forms  a  superficial  film. 

9.  Heat  again,  without  stirring,  on  a  piece  of  asbestos 
over  the  flame.     A  film  will  gradually  form  and  harden 
with  a  dry  surface. 

10.  Now  adjust  the  weight  by  calculating  the  total 


i4       LABORATORY   GUIDE   IN   BACTERIOLOGY 


weight  of  the  different  ingredients,  allowing  1000  g.  for 
all  the  water  used  and  30  g.  for  the  weight  of  the  white  of 
each  egg  and  the  weight  of  the  pan.  If  the  actual  total 
weight  is  in  excess  of  the  amount  calculated,  boil  again 


—  a 


FIG.  5 

Apparatus  for  Filtering 
Media 

a.  Filter 

b.  Large  funnel 

c.  Small  funnel 

d.  Rubber  hose 

e.  Pinchcock 
/.  Pipette 

g.  Culture-tube 


until  the  proper  weight  is  reached.  If,  on  the  other  hand, 
the  total  weight  is  less,  make  up  the  deficiency  by  adding 
water. 

Example — 

TVater 

Agar 

Extract  of  beef 

Peptone 

Whites  of  two  eggs 

Saucepan  for  example  . . 


3000.  O  

.100.00$ 

15-0  

...1.50 

3-°  

...  o  .  30 

IO.O  

...  I  .  00 

60.0 

450.0 

Total. 


PREPARATION  OF  CULTURE  MEDIA 


By  placing  weights  to  correspond  to  this  amount  on  the 
left  side  of  the  scales,  and  the  saucepan  with  contents 
on  the  other  side,  the  weight  can  be  readily  adjusted. 

ii.  Heat  again  to  the  boiling-point,  and  filter  the 
liquid  into  culture-tubes  to  about  one-third  of  their 
length  (7  c.c.). 

Method  of  filtering  media. — Arrange  two  funnels,  a 
pipette  with  hose,  and  a  pinchcock  on  the  retort-stand, 
as  illustrated  in  Fig.  5.  Place  a  strainer  made  of  per- 
forated tin  (Fig.  6)  against  the  side  of  the  saucepan, 


FIG.  7 
Strainer  in  Position 

near  the  top,  as  illustrated  in  Fig.  7,  and  pour  the  medium 
into  the  larger  funnel  holding  the  filter. 

For  the  purpose  of  filtering  the  medium  either  white 
filter-paper  of  the  best  quality  or  absorbent  cotton  may 
be  used.  A  filter-paper  folded  in  the  following  manner 
is  very  serviceable: 

i.   Take  a  square  piece  of  filter-paper  twice  as  wide 


16       LABORATORY   GUIDE   IN   BACTERIOLOGY 

as  the  depth  of  the  funnel  to  be  used  (Fig.  8,  a),  and  fold 
to  half  the  size  (Fig.  8,  b)  so  as  to  make  i  cover  2,  and  3 
cover  4 

2.  Fold  this  to  make  i  cover  2  and  3  cover  4  (result: 
Fig.  8,  c).     It  consists  of  four  layers  and  forms  a  square. 

3.  Fold  the  upper  part,   consisting  of  two  layers, 


a- 


FIG.  8 

Method  of  Folding  Paper  Filter 
(For  reference  letters  see  text) 


from  i  to  2  (Fig.  8,  d).     The  shaded  triangle,  2-3-4, 
now  has  six  layers;  the  other  1-3-4,  two  layers. 

4.   Fold  the  upper  double  layer  so  as  to  make  2  cover 
a  point  in  the  diagonal  at  5,  taking  care  to  make  a  sharp 


PREPARATION    OF   CULTURE   MEDIA  17 

point  at  4  (result:  Fig.  8,  e).     The  shaded  part  is  now 
eight  layers  deep. 

5.  Turn  the  folder  face  down,  and  repeat  the  opera- 
tions exactly  as  in  3  and  4  (result:   Fig.  8,  /). 

6.  Take  up  and  open  the  large  middle  fold  (result: 
Fig.  8,  g).     The  two  halves  must  now  be  symmetrical. 

7.  Fold  so  as  to  make  the  lines  1-3  and  1-4  meet 
at  the  center  line  1-2  (result:   Fig.  8,  h). 

8.  Now  pick  up  and  fold  backward  so  as  to  have  i 
cover  2  in  the  back  (result:   Fig.  8,  i). 

9.  Cut  through   the   line    1-2   and  open  up.     The 
extreme  ends  will  be  found  without  a  fold,  and  may  be 
folded  so  as  to  make  nine  sharp  edges. 

This  filter  is  then  evenly  inserted  into  the  funnel, 
spreading  the  folds  at  a  distance 
from  each  other  as  nearly  alike 
as  possible.  Great  care  should  be 
taken  to  make  the  folds  and  the 
point  sharp,  as  this  insures  rapid 
filtering  of  the  liquid  and  prevents 
the  filter  from  tearing.  A  wire 
rack  may  be  used,  but  does  not  FIG.  9 

yield  nearly  so  good  results  (Fig.  9). 

Another  method,  simple  and  yielding  excellent  results, 
is  the  use  of  absorbent  cotton.  Take  a  piece  about  3 
inches  square,  and  cover  lightly  the  lower  end  of  the 
funnel  with  it.  Take  a  larger  piece,  also  square,  judging 
the  size  according  to  the  size  of  the  funhel;  pull  it  gently, 
so  as  to  make  the  layer  of  cotton  thinner,  without  showing 
any  open  spaces;  and  spread  this  around  the  inner  sur- 
face of  the  funnel. 

If  a  vacuum  pump  is  available,  the  medium  may  be 


i8       LABORATORY   GUIDE    IN    BACTERIOLOGY 

filtered  rapidly  by  the  use  of  suitable  apparatus,  as  illus- 
trated in  Fig.  10.  At  the  connection  with  the  vacuum 
pump  a  valve  should  be  inserted — or  a  flask  with  a  rub- 


FIG.  10 
Filtering  Media  by  Means  of  Vacuum  Pump 

a.  Liquid  medium  e.  Reflex  flask 

b.  Absorbent  cotton  /.  Rubber  stopper  with  two  holes 

c.  Rubber  stopper  g.  Connection  with  aspirator 

d.  Filtered  medium 

ber  cork  with  two  holes  may  take  its  place — to  prevent 
the  water  from  entering  the  flask. 

Whichever  method  is  employed,  the  filter  must  always 
first  be  soaked  in  hot  water,  or,  better,  kept  in  steam 
until  used. 


PREPARATION    OF   CULTURE   MEDIA  19 

The  filtered  liquid  is  collected  in  a  smaller  funnel 
(Fig.  5,  c).  A  wire  basket  should  be  supported  so  as  to 
be  inclined  to  an  angle  of  about  45°,  so  as  to  allow  the 
proper  arrangement  of  the  tubes. 

Remove  the  plug  of  a  culture-tube  and  quickly  pass 
the  latter  up  the  full  length  of  the  pipette  (Fig.  5,  /),  which 
should  be  no  less  than  3  inches  long.  Open  the  pinch- 
cock,  fill  the  tube  one-third  full,  and  then  quickly  with- 
draw the  tube  so  as  to 
leave  no  trace  of  the  me- 
dium on  the  upper  part  of 
the  tube,  and  replace  the 
cotton  stopper.  If  this 
precaution  is  neglected, 
the  cotton  stopper  will  stick 
to  the  glass,  which  will 
cause  a  great  deal  of  an- 
noyance later  on  and  ex- 
pose the  medium  to  the 
danger  of  contamination. 
Fill  thirty  tubes  in  this 
manner. 

If  an  exact   volume  of 
medium  is   required   in  a 
test-tube,    an    apparatus 
such  as  is  shown  in  Fig.  FlG-  " 

.  Filling  Definite  Amount  of  Medium 

ii  may  be  employed.  into  Culture-Tube 

EXERCISE  II.   PREPARATION  OF  GLUCOSE-AGAR 

Glucose-agar  is  especially  used  for  the  demonstration 
of  gas-forming  organisms.  In  such  cases  the  glucose  is 
decomposed,  and  gas  appears  in  bubbles  throughout  the 
medium. 


20       LABORATORY   GUIDE   IN   BACTERIOLOGY 

Preparation — 

1.  Note  the  weight  of  an  Erlenmeyer  flask  (500  c  c.). 

2.  Weigh  3.75  (ij  per  cent.)  g.  of  glucose  into  the 
flask. 

3.  Filter  250  g.  of  plain  agar  into  the  flask,  heat,  and 
agitate  until  the  glucose  is  completely  dissolved. 

4.  Fill  twenty  tubes  in  the  same  manner  as  above. 

NOTE. — By  tying  a  string  across  a  wire  basket  near  the  top, 
the  plain  agar-tubes  may  be  kept  separate  from  the  glucose-agar. 
A  slip  of  paper  indicating  the  medium  and  the  date  of  its  prepara- 
tion must  always  be  inserted.  These  two  media  are  very  diffi- 
cult to  distinguish  by  the  eye,  and  failure  to  label  them  properly 
will  lead  to  entirely  unreliable  results. 

The  media  should  now  be  sterilized  in  the  autoclav. 
The  principle  of  this  mode  of  sterilization  is  the  applica- 
tion of  steam  under  pressure.  Certain  bacteria,  and 
some  of  these  are  very  widely  distributed  in  nature,  have 
the  faculty  of  forming  spores.  These  spores  are  very 
highly  resistant  to  heat  and  do  not  lose  their  vitality  either 
by  boiling  or  by  application  of  heat  under  ordinary  atmos- 
pheric pressure,  although  all  vegetative  forms  are  killed. 
By  adding  the  pressure  of  one  atmosphere  to  ordinary 
pressure,  the  boiling-point  is  raised  to  121.40°,  which  is 
sufficient  to  kill  all  spores  during  an  exposure  of  5 
minutes;  media  in  flasks  should  be  given  10  minutes. 

The  autoclav  consists  of  a  strong  cylinder,  made  of 
iron,  with  a  bottom  and  removable  lid.  Inside  is  a 
basket,  or  rack  with  shelves,  resting  on  three  short  sup- 
ports with  a  centrally  located  hole  in  the  bottom.  The 
lid  fits  closely  (in  some  autoclavs  with  a  large  washer), 
and  is  provided  with  a  steam-valve  (Fig.  12,  a),  a  safety- 
valve  (Fig.  12,  b),  and  a  gauge  (Fig.  12,  c).  The  latter 
indicates  the  pressure  and  temperature.  The  top  is 


PREPARATION   OF  CULTURE   MEDIA 


21 


fastened  by  means  of  thumbscrews  (Fig.  12,  d-d).  The 
heat  is  produced  by  two  circular  sets  of  Bunsen  burners 
in  a  hollow  cylinder  at  the  bottom  (Fig.  12,  e). 

Other  forms  based  on  the  same  principles  are  also 
in  use. 

Before  using  the  autoclav  the  inside  should  be  exam- 


Pic.  12 
Autoclav 

a.  Steam-valve 

b.  Safety-valve 

c.  Gauge 

d-d.  Thumbscrews 

e.  Bunsen    burner    and 
opening 


ined.  It  must  be  clean,  and  there  must  be  enough  clean 
water  in  it  to  reach  nearly  to  the  bottom  of  the  basket. 
Water  containing  large  amounts  of  impurities  is  liable  to 
foam  when  boiling,  and  thus  wet  the  plugs  and  ruin  the 


22       LABORATORY   GUIDE   IN   BACTERIOLOGY 

medium.  Place  the  material  to  be  sterilized  in  the  basket. 
Fit  the  lid  of  the  autoclav  by  joining  corresponding 
marks  (usually  a  number  or  a  letter)  on  the  side  of  the 
lid  and  margin  of  the  main  body.  Then  tighten  the 
thumbscrews  by  hand,  always  tightening  those  diamet- 
rically opposite  to  each  other  at  the  same  time.  See  that 
the  steam-valve  is  open,  and  then  light  the  gas.  Regu- 
late the  safety-valve  to  blow  off  at  the  required  pressure, 
if  provided  with  the  means  of  doing  this. 

It  is  necessary  to  allow  the  steam  to  escape  for  about 
one  minute,  in  order  to  drive  all  air  out  of  the  cylinder. 
Then  close  the  steam-valve.  The  pressure  will  begin  to 
increase,  as  indicated  by  the  hand  of  the  manometer. 
When  the  desired  temperature  is  reached,  shut  off  the 
gas  from  the  outer  ring  of  burners  and  regulate  the  gas 
pressure  of  the  inner  rings,  to  maintain  the  proper  tem- 
perature. It  is  sufficient  to  expose  the  media  to  a  tem- 
perature of  120°  for  5  minutes.  After  this  has  been 
accomplished,  shut  off  the  gas  and  allow  to  cool  to 
normal  pressure,  or  100°  or  below.  The  lid  should  not 
be  released  before  this,  as  a  sudden  diminution  of  pressure 
causes  the  contents  of  the  tubes  suddenly  to  boil  up  and 
push  the  stoppers  out. 

The  steam-valve  should  be  opened  when  the  auto- 
clav has  cooled  to  100°  or  below.  This  will  guard 
against  the  possibility  of  the  top  blowing  off  and  injuring 
the  operator,  if  the  gauge  does  not  register  properly. 
On  the  other  hand,  the  suction  caused  by  the  contrac- 
tion of  the  steam  may  hold  the  lid  down,  so  that  it  cannot 
be  removed  until  the  pressure  is  equalized. 

When  sterilizing  media  which  contain  carbohydrates 
(glucose-agar,  for  instance),  it  is  important  not  to  allow 


PREPARATION   OF   CULTURE   MEDIA  23 

the  temperature  to  rise  beyond  120°,  nor  to  allow  it  to 
remain  at  that  point  any  longer  than  5  minutes.  Car- 
bohydrates are  easily  broken  up  by  heat.  They  are 
then  valueless  in  the  medium,  and  also  generate  an  acid 
reaction  as  a  result  of  chemical  decomposition. 

In  the  preparation  of  agar  more  difficulties  are  met 
with  than  in  the  preparation  of  any  other  medium.  The 
main  difficulty  is  the  fact  that  it  takes  a  long  time — from 
30  to  45  minutes — for  the  agar  to  dissolve,  and  this 
creates  the  danger  of  burning  it  by  overheating.  Great 
care  mus't  therefore  be  exercised  in  the  preparation 
at  all  stages. 

EXERCISE  III.      PREPARATION   OF   PEPTONE  GELATIN 

1.  Weigh    the    saucepan    and    measure   35oc.c.   of 
tap-water  into  it.     This  amount  includes  50  c.c.  allow- 
ance for  evaporation. 

2.  Dissolve  0.75  g.  of  extract  of  meat,  and,  when  near 
boiling,  add  3  g.  of  peptone. 

3.  When  boiling,  add  during  the  cold  season  10  per 
cent.  (30  g.),  during  the  warm  season  12  per  cent.  (36  g.), 
of  the   best  gelatin    (Gold  label)  by  dissolving  two  or 
three  leaves  at  a  time,  with  constant  stirring. 

4.  When  completely  dissolved,  adjust  the  reaction 
as  directed  in  the  preparation  of  agar.     As  gelatin  con- 
tains considerable  acid,  it  will  take  mere  NaOH  solu- 
tion in  proportion  than  for  agar. 

5.  Then  cool  to  60°,  and  stir  into  the  mixture  the 
white  of  one  egg  dissolved  in  30  c.c.  of  water.     Slowly 
heat  over  the  flame  on  a  piece  of  asbestos,  without  stir- 
ring, until  the  egg  albumen  is  completely  coagulated  and 
forms  a  dry  film  on  top. 


24       LABORATORY   GUIDE   IN   BACTERIOLOGY 


6.  Adjust  the  weight,  and  filter  through  paper  or 
absorbent  cotton  previously  moistened  with  hot  water, 
and  distribute  into  thirty  culture-tubes. 

7.  Sterilize  in  the  autoclav  for  5  minutes  at  120°. 
Care  is  necessary  not  to  overheat  this  medium,  because 
gelatin  easily  loses  the  property  of  solidifying  when  cool. 
In  order  to  avoid  this,  the  discontinuous  or  intermittent 
method  of  sterilization  is  often  used.     By  this  method 
the  medium  is  exposed  in  a  steam  sterilizer  to  steam  at 
the  temperature  of  100°  for  20  minutes  on  three  con- 
secutive days.     The  first  day  all  vegetative  forms  are 


FIG.  13  FIG.  14 

Arnold  Steam  Sterilizer  \  Arnold  Steam  Sterilizer 

The  hood  is  taken  off  and  the  door       \  a.  Inner  water  compartment 

opened,  showing  inside  arrangement  b.  Outer  water  compartment 

c.  Perforated  bottom 

d.  Sterilizing  chamber 

e.  Sheet-copper  walls 

killed.  By  keeping  the  medium  at  room  or  incubator 
temperature,  spores,  which  may  be  present,  will  develop 
into  the  vegetative  form  and  be  killed  by  the  second  day's 
exposure.  If  after  this  any  spores  should  happen  to 


PREPARATION   OF   CULTURE   MEDIA  25 

survive,  they  will  develop  during  the  next  24  hours,  and 
the  third  exposure  to  steam  will  complete  the  sterilization. 
The  apparatus  used  for  this  purpose  is  the  "Arnold  Steam 
Sterilizer."  The  usual  form  used  is  seen  in  Figs.  13  and 
14.  Fig.  13  illustrates  the  appearance  of  the  ordi- 
nary form  with  the  hood  off.  Fig.  14  shows  the  inside 
arrangement,  a  and  b  being  two  compartments  connected 
by  small  holes,  and  in  which  a  certain  amount  of  water 
has  to  be  kept.  The  water  contained  in  the  inner  com- 
partment (a)  is  brought  to  a  boil  by  a  Eunsen  burner, 
the  steam  rising  through  a  number  of  holes  in  the  bottom 
(c)  into  the  chamber  (d).  The  steam  condenses  at  the 
top  of  the  chamber  and  returns  between  two  sheet-copper 
walls  (e,  e)  to  the  large  compartment  (b).  A  larger  form 
of  steam  sterilizer,  constructed  on  the  same  principle,  is 
convenient  for  sterilizing  large  amounts  of  the  media. 

EXERCISE  IV.    PREPARATION  OF  PEPTONE-BROTH  (FRENCH : 
BOUILLON)  AND  DUNHAM'S  PEPTONE-SOLUTION 

1 .  Weigh  the  saucepan  and  measure  600  c.c.  of  tap- 
water  into  it  and  heat. 

2.  Dissolve,  when  hot  (but  not  boiling),  6  g.  of  pep- 
tone. 

3.  When    completely  dissolved,  replace   the   evapo- 
rated water  and  divide  into  two  equal  amounts  (300  c.c. 
each). 

4.  Filter   one   part  twice    through  the   same   filter 
(paper)  and  distribute  into  twenty-five  tubes.     Sterilize 
in  autoclav  for  5  minutes  at  120°.     This  is  Dunham's 
peptone-solution. 

5.  Dissolve  completely  i  g.  of  extract  of  beef  in  the 
other  300  c.c. 


26       LABORATORY   GUIDE   IN   BACTERIOLOGY 


6.  Adjust  the  reaction  in  the  same  manner  as  above. 

7.  Fill  into  an  Erlenmeyer  flask   and   autoclav  for 
10  minutes  at  120°. 

8.  Keep  the  Erlenmeyer  flask  and  contents  for  24 
hours  in   a  cool  place,  filter,  and  then  distribute  into 


FIG.  16 

Potato  Cylinder 
showing  diagonal  for  cutting 

twenty-five  tubes,  and  autoclav  these  at  1 20°  for  5  min- 
utes.    This  is  "peptone-broth." 

NOTE. — The  reason  for  exposing  broth  to  a  heat  of  120° 
twice  is  this:  The  solution  contains  a  considerable  quantity  of 
substances,  which  are  precipitated  by  heat  and  appear  as  a  sedi- 
ment after  cooling.  As  it  is  important  to  have  a  perfectly  clear 
broth  in  tubes,  these  substances  are  precipitated  by  the  first 
heating,  and,  if  tubed  later,  the  second  sterilization  will  not 
affect  the  appearance  of  the  medium. 

Broth  was  originally  prepared  from  chopped  beef, 
and  for  many  purposes  this  is  preferable.  The  method 
of  preparation  is  as  follows: 


PREPARATION   OF   CULTURE   MEDIA  27 

1.  500  g.  (i  pound)  of  lean  minced  beef,  as  free  as 
possible  from  fat  and  tissues,  is  added  to  1000  c.c.  of 
tap-water  and  set  aside  on  ice  for  24  hours. 

2.  Weigh  a  saucepan,  and  cook  the  beef  and  water 
for  about  one  hour. 

3.  Strain   through   cheese-cloth,   and   press  all   the 
liquid  out  in  a  meat-press  (Fig.  15). 

4.  Replace  the  water  lost  by  evaporation. 

5.  Dissolve  10  g.  of  Witte's  peptone. 

6.  Adjust  the  reaction. 

7.  Filter  into  flasks,  and  sterilize  in  autoclav  at  120° 
for  10  minutes.     It  may  then  be  distributed  into  culture- 
tubes. 

Fresh  meat,  as  well  as  extract  of  meat,  often  contains 
a  small  amount  of  muscle-sugar  (glycogen).  If  it  is 
necessary  to  prepare  a  sugar-free  broth,  a  pure  culture 
of  Bacillus  coli  is  added  to  the  mixture  of  chopped  beef 
and  water,  and  this  is  incubated  at  37°  for  24  hours. 
After  this  the  preparation  is  the  same  as  described. 

EXERCISE  V.      PREPARATION  OF  POTATO 

1.  Select  several  large  potatoes,  and  cleanse  by  brush- 
ing all  dirt  off  carefully  and  washing  in  water. 

2.  Punch  out  cylinders  with  a  borer  of  suitable  size, 
trim  them,  and  cut  them  into  two  equal  parts  (Fig.  16). 
Cut  with  a  sharp  knife  along  the  diagonal. 

3.  Immerse  the  pieces  in  running  water  for  24  hours. 

4.  Insert   one   half -cylinder   into   each   potato-tube 
so  that  the  wide  end  rests  on  the  constriction,  then  pour 
a  small  amount  of  water  into  it.     If  the  potato-tubes 
have  no  constriction,  place  a  small  wad  of  cotton  in  the 
bottom  and  moisten  this  with  water  (Figs.  17  and  18). 


28       LABORATORY   GUIDE   IN   BACTERIOLOGY 


5.  Sterilize  in  autoclav  for  5  minutes  at  120°,  or  in 
Arnold  for  3  consecutive  days  with  20-30  minutes' 
exposure. 

EXERCISE  VI.      PREPARATION   OF   LITMUS   MILK 

i.   Separate  five-sixths  of  the  cream  from  the  milk. 


FIG.  17  FIG.  18 

Ordinary  Style  of  Potato-Tube  Potato-Tube 

a.  Potato        b.  Cotton 

2.  Add  7-10  per  cent,  of  a  solution  of  litmus  (tincture 
of  litmus). 

3.  Distribute  about  yc.c.  into  each  of  twenty-five  tubes. 

4.  Sterilize  in  autoclav  for  5  minutes  at  120°,  or  in 
Arnold  for  3  consecutive  days. 

NOTE. — The  prepared  culture  media  should   be   carefully 
stored  in  a  dark,  cool  place.     If  they  are  to  be  kept  for  a  consid- 


PREPARATION   OF   CULTURE  MEDIA  29 

erable  length  of  time,  they  should  be  sealed  either  with  paraffin 
or  with  a  rubber  cap.  They  should  always  be  protected  from 
dampness,  as  mold  fungi  are  apt  to  alight  on  the  cotton  stoppers 
and  send  their  filaments  into  the  tube.  Before  inoculation,  they 
should  always  be  carefully  examined,  and  those  which  show 
cloudiness  or  colonies,  as  well  as  those  which  have  shrunk  from 
evaporation,  should  be  rejected. 


CHAPTER  IV 

PREPARATION  OF  STAINING  SOLUTIONS 
Saturated  alcoholic  solutions  of  stains  are  prepared 
by  covering  an  arbitrary  amount  of  stain  with  absolute 
alcohol.  The  solution  is  saturated  as  long  as  any  of  the 
stain  remains  undissolved  at  the  bottom  of  the  vessel. 
Other  solutions  necessary  for  the  preparation  of  stains  are: 

1.  Solution  of  potassium  hydrate  in  water  i :  10,000. 

2.  Solution  of  carbolic  acid  in  water  (5  per  cent.). 

3.  Anilin- water,  prepared  by  shaking  anilin-oil  with 
water  (2  per  cent.)  and  filtering  twice  through  the  same 
paper.     It  should  be  perfectly  clear. 

The  staining-bottles  usually  employed  have  a  capaci- 
ty of  about  30  c.c.  The  following  amounts  will  nearly 
fill  them: 

1.  Loeffler's  methylene-blue : 

Saturated  alcoholic  solution  of  methylene-blue  9  c.c. 
Potassium  hydrate  in  distilled  water  i :  10,000  21  c.c. 

2.  Ziehl-Neelsen's  carbol-f uchsin : 

Saturated  alcoholic  solution  of  f uchsin 3  c.c. 

5$  carbolic  acid  solution  in  water 27  c.c. 

3.  Ehrlich's  anilin-gentian- violet : 

Saturated  alcoholic  solution  of  gentian-violet  7.5  c.c. 

Anilin-water 22.5  c.c. 

This  stain  requires  occasional  filtering  and  is  somewhat 
unstable. 

4.  Gram's  iodin  solution: 

lodin o.i  g. 

Potassium  iodid 0.2  g. 

Dissolve  in  about  2  c.c.  of  water,  and  then  add  enough 
water  to  make  the  total  measure  30  c.c. 

3° 


CHAPTER  V 
THE  MICROSCOPE 

(Fig.  19) 

The  compound  microscope  is  a  necessary  adjunct  to 
any  kind  of  bacteriological  work.  For  this  work  three 
objectives  (Leitz  No.  3,  No.  6,  or  No.  7  and  fa  oil  im- 
mersion, Zeiss  No.  A  or  No.  AA,  No.  D  or  No.  DD,  and 
•fa  oil  immersion)  and  two  oculars  (Nos.  2  and  4)  are 
indispensable.  For  the  intelligent  manipulation  of  the 
microscope  it  is  absolutely  necessary  to  understand  the 
underlying  optical  principles,  which  may  be  studied  from 
special  works  on  the  subject. 

References — 

S.  H.  Gage,  The  Microscope. 

Carpenter  and  Dallinger,  The  Microscope  and  its  Revelations. 

For  use  in  the  laboratory  it  will  be  sufficient  to  call 
attention  to  some  of  the  most  important  points  to  be 
observed. 

The  usual  pattern  of  microscope  consists  of  two  main 
parts:  the  stand,  and  the  optical  parts  (Fig.  19)  which 
are  attached  to  the  stand. 

The  stand  consists  of  a  body-tube,  draw-tube,  coarse 
adjustment,  fine  adjustment  (micrometer  screw)  in  a 
pillar,  nose-piece,  stage  with  clips  for  holding  the  object, 
main  pillar,  and  the  horseshoe  base.  At  the  junction 
of  the  main  pillar  and  the  fine-adjustment  pillar  is  the 
inclination  joint. 

The  draw- tube,  regulating  the  focal  length,  which 
varies  in  different  instruments,  should  be  raised  to  16 


a 


e 


FIG. 19 
Microscope  (after  E.  Leitz) 


a.  Ocular 

b.  Place  where    virtual    picture 

formed 

c.  Body-tube 

d.  Coarse  adjustment 

e.  Micrometer  screw 
/.  Inclination  joint 
g.  Horseshoe  base 
h.  Mirror 


*.   Iris  Diaphragm  and  Condenser 

k.  Objective 

I.    Nose-piece 

m-o.  Picture  as  it  appears  to    the  eye 

p.-g.  Object  (cover-slip) 

r.  Adjustment  spring 

s.  Stage 

/.  Object  (slide) 


THE   MICROSCOPE 


33 


or  17  mm.  If  a  nose-piece  is  attached,  the  width  of 
this  must  be  deducted  from  the  tube-length. 

The  optical  parts  are  the  oculars,  the  objectives,  the 
substage  condenser,  and  the  mirror.  The  ocular  is  a 
combination  of  lenses,  which  slips  into  the  top  of  the 
draw-tube  and  is  nearest  the  eye.  The  objective  is  a 
combination  of  lenses  which  is  screwed  into  the  nose- 
piece  and  fits  to  the  lower  end  of  the  draw-tube.  The 
substage  condenser  fits  under  the  stage.  It  concentrates 
the  light  on  the  object  and  is  raised  for  high  powers  or 
lowered  for  low  powers.  At  the  lower  end  of  this  con- 
denser is  the  iris  diaphragm,  which  is  regulated  by  a 
small  lever  with  a  milled  head,  and  serves  the  purpose 
of  regulating  the  light  supply.  The  mirror  has  two 
sides,  a  concave  and  a  flat. 

In  the  manipulation  of  the  compound  microscope  the 
following  points  should  be  strictly  observed: 

1.  Keep  the  instrument  scrupulously  clean.     When 
not  in  use,  lock  it  in  the  case  or  cover  it  with  a  bell-jar. 

2.  When  carrying  the  instrument,  grasp  it  by  the 
main  pillar  underneath  the  stage,  not  by  the  fine-adjust- 
ment pillar.     The  fine  adjustment   consists   of   a   very 
delicate  screw-thread,  which  is  easily  damaged. 

3.  The  lenses,  condenser,  and  mirror,  when  needing, 
should  be  wiped  with  Japanese  lens-paper,  never  with 
any  coarse  material. 

4.  For  cleaning  use  a  damp  cloth.     For  wiping  the 
lenses  use  water  or  xylol.     Never  use  alcohol,  as  this 
dissolves  the  cement  holding  the  lenses  in  place  and  also 
injures  the  lacquer. 

5.  Do  not  take  the  instrument  apart.     The  working 
parts  are  of  extremely  delicate  nature  and  easily  injured. 


34       LABORATORY   GUIDE   IN   BACTERIOLOGY 

Be    careful    not    to   drop    the    oculars    or    objectives. 

6.  The  inclination  joint  can  be  used  only  with  dry 
lenses  and  dry  objects,  not  with  the  oil-immersion  or 
hanging-drop  preparations. 

7.  After  placing  the  object  on  the  stage,  focus  with 
a  low  power  and  the  coarse  adjustment.     With  high 
powers  use  the  coarse  adjustment  first  and  the  fine  adjust- 
ment afterward.     The  free  use  of  the  fine  adjustment 
saves  the  accommodation  of  the  eye.     As  the  eye  is  cap- 
able of  accommodating  itself  to  distances,  it  may  with  an 
effort  distinguish  a  picture  which  is  not  in  perfect  focus. 
This  effort  is  saved  by  using  the  fine  adjustment. 

8.  Always  raise  the  draw-tube  by  means  of  the  coarse 
adjustment  before  changing  the  objectives  or  examining 
a  different  preparation. 

9.  Before  focusing,  obtain  as  good  a  light  as  possible 
by  turning  the  mirror,  and  then  regulate  the  supply  by 
the  diaphragm.     Always  use  reflected  light,  never  direct 
sunlight. 

10.  When  focusing  an  object,  lower  the  draw-tube 
until  the  lens  almost  touches  the  cover-glass.     This  can 
easily  be  seen  by  looking  at  the  instrument  from  one  side 
and  watching  the  reflection  of  the  objective  in  the  cover- 
glass.     Then,  with  the  eye  at  the  ocular,  slowly  focus  up. 
Do  not  focus  down  with  the  eye  at  the  ocular,  as  the  lens 
may  then  come  into  violent  contact  with  the  object, 
destroying  the  latter  and  injuring  the  lens. 

n.  For  living  or  transparent  objects  use  as  little 
light  as  possible.  For  stained  or  opaque  objects  more 
light  is  necessary. 

12.  Do  not  use  higher  powers  than  is  necessary. 

13.  To  use  the  oil-immersion  lens,  place  a  drop  of 


THE   MICROSCOPE  35 

clear  cedar  oil,  free  from  dust  and  air-bubbles,  on  the 
cover-glass,  which  must  be  perfectly  dry.  In  this  case, 
by  careful  manipulation,  the  objective,  after  being 
brought  in  touch  with  the  oil  by  means  of  the  coarse 
adjustment,  may  be  gradually  lowered  by  the  fine  adjust- 
ment until  the  object  is  focused;  or  lower  the  objective 
until  almost  in  touch  with  the  cover-glass,  and  focus  up. 
High  powers  always  require  the  use  of  a  homogeneous 
liquid  between  the  cover-glass  and  the  front  lens  of  the 
objective,  to  avoid  loss  of  light  by  refraction.  As  a  bundle 
of  rays  disperses  when  entering  a  thinner  medium  from  a 
denser  one,  there  is  not  sufficient  light  entering  the  objec- 
tive to  make  objects  discernible,  when  using  high  powers. 
By  the  insertion  of  a  liquid  (inspissated  oil  of  cedar)  of 
the  same,  or  nearly  the  same,  refractive  index,  a  homo- 
geneous connection  is  established  between  the  cover-glass 
and  the  objective,  thus  avoiding  this  loss  and  allowing  a 
bundle  of  light  of  sufficient  power  to  enter  the  objective.1 

14.  After  using  the  oil-immersion  lens,  wipe  the  oil 
off  with  lens-paper.     If  the  oil  sticks  to  the  lens,  wipe  it 
off  with  xylol,  never  with  alcohol.     At  the  same  time 
wipe  the  oil  off  the  cover-slip. 

15.  The  microscope  should  stand  on  a  firm  table, 
to  avoid  being  shaken.     The  table  should  be  low  enough 
so  as  not  to  necessitate  bending  the  body. 

1 6.  Always  keep  both  eyes  open.    This  saves  the 
eyesight.     Beginners  find  this  a  difficult  rule  to  apply, 
but  with  very  little  practice  and  persistence  it  is  easily 
accomplished.     Also  use  both  eyes  alternately. 

17.  It  is  always  well  to  move  the  object  while  bringing 

'For  detailed  description  and  diagrams  see  S.  H.  Gage  The 
Microscope. 


36      LABORATORY   GUIDE   IN   BACTERIOLOGY 

it  into  focus.  It  is  then  easy  to  feel  if  the  lens  touches 
the  glass,  and  a  moving  object  is  seen  much  more  easily 
than  a  stationary  one. 

1 8.  Use  the  plain  mirror  in  combination  with  the 
condenser,  and  the  concave  mirror  without  the  condenser 
or  with  artificial  light. 

19.  In  preparing  stained  preparations,  it  often  hap- 
pens that  a  small  amount  of  stain  remains  on  the  upper 
side  of  the  cover-slip.     Care  must  be  taken  to  focus  for 
a  plane  below  this. 


CHAPTER  VI 
COLLECTING  BACTERIA  FROM  THE  AIR 

1.  Sterilize  all  Petri  dishes  in  the  hot-air  sterilizer  for 
one  hour  at  160°. 

2.  Melt  two  tubes  of  plain  agar  and  one  of  glucose- 
agar  in  the  water-bath.     The  water-bath  (Fig.  20)  is  a 
round  copper  vessel  with  a  number  of  holes  in  the  top. 


These  holes  are  large  enough  to  allow  a  culture-tube  to 
slip  in  easily.  A  thermometer  is  passed  through  a  rubber 
cork  with  a  hole  in  its  center,  and  inserted  into  one 
of  the  holes  in  the  water-bath.  Enough  water  is  then 
poured  into  the  apparatus  to  fill  it  about  two-thirds,  and 
the  thermometer  is  lowered  until  the  mercury  bulb  is 

37 


38      LABORATORY  GUIDE  IN  BACTERIOLOGY 

completely  immersed  in  the  water.     The  culture-tubes 
are  then  slipped  in,  and  the  water  is  heated  to  100°. 

3.  Singe  the  cotton  stopper  of  the  liquefied  agar- 
tubes  in  the  flame,  remove  the  cotton  stopper,  pass  the 
mouth  and  about  one  inch  of  the  tube  through  the  flame, 
and  pour  the  contents  into  a  sterile  Petri  dish,  carefully 
lifting  the  cover  (Fig.  21)  and  quickly  replacing  it. 

4.  Repeat  this  operation  with  the  second  agar-tube. 

5.  Place  both  Petri  dishes  containing  the  liquid  agar 
on  a  level  surface. 

6.  When  the  agar  is  solidified,  expose,  by  removing 
the  cover,  one  dish  to  the  air  of  the  laboratory,  and  the 
other  outside  on  the  window-sill,  for  10  minutes. 


FIG.  21 
Pouring  Medium  into  Petri  Dish 

7.  Replace  the  cover  and  place  in  lockers. 

8.  Cool  the  water-bath  to  43°  exactly,  and  mix  the 
scrapings  from  under  a  finger  nail  with  the  liquid  glucose- 
agar.     Keep  this  also  in  the  locker. 

9.  Remove  the  plug  of  a  tube  of  broth  and  place  a 
hair  in  the  liquid.     Keep  also  in  the  locker. 

When  inoculating  liquefied  agar  media,  it  is  necessary 
to  do  so  at  a  temperature  no  higher  than  43°  nor  lower 
than  40°.  Above  43°  the  organisms  are  liable  to  be 
injured  by  heat;  below  40°  the  agar  solidifies,  and  an 
even  distribution  is  impossible.  If  gelatin  is  used,  the 
latter  precaution  is  not  imperative,  as  gelatin  solidifies  at 


COLLECTING  BACTERIA  FROM  THE  AIR       39 

about  25°.  Observe  and  make  notes  on  the  appearance 
of  these  Petri  dishes  after  24  hours.  By  this  time  it  will 
be  observed  that  a  number  of  spots  of  different  sizes, 
shapes,  colors,  etc.,  have  formed  on  the  surface  of  the 
medium.  Each  of  these  spots  probably  consists  of  a 
large  number  of  the  same  species  of  organism,  and  is 
called  a  colony.  Besides  bacteria,  yeasts  and  mold  spores 
are  constantly  present  in  the  atmosphere  and  drop  on  the 
plate.  These  organisms,  being  of  higher  specific  gravity 
than  the  air,  are  constantly  falling,  or  are  carried  hither 
and  thither  by  currents  in  the  atmosphere.  Small 
particles  of  dust  also  may  alight  on  the  medium,  and  then 
form  a  dark  spot  near  the  center  of  the  colony.  Dust 
particles  are  usually  covered  with  bacteria,  possibly  of 
different  species,  and  in  this  case  the  resulting  colony  may 
not  be  composed  of  one  and  the  same  species.  These 
are  not  "pure  cultures." 

Inoculate  two  or  three  colonies  on  agar-slants. 

NOTE. — Agar-slants  are  prepared  by  allowing  liquid  agar  to 
solidify  in  a  slanting  position. 

Method  oj  inoculation — 

1.  Singe  the  cotton  plug  of  a  tube  containing  the 
medium.     It  is  imperative  always  to  singe  the  cotton 
plugs  of  tubes  before  opening  them,  whether  these  tubes 
contain  a  culture  or  a  sterile  medium.     The  organisms 
from  the  air  are  constantly  falling  on  the  cotton,  and 
bacteria  may  also  be  deposited  on  the  cotton  by  handling 
it  with  the  fingers.     If  these  organisms  were  not  killed  by 
the  process  of  singeing,  they  would  drop  on  the  medium 
after  removal  of  the  stopper,  and  thus  ruin  a  pure  culture. 

2.  Hold  the  tube  (or,  if  a  transfer  is  made,  both  tubes 
side  by  side)  between  the  thumb  and  the  forefinger,  so 


40      LABORATORY  GUIDE  IN  BACTERIOLOGY 

that  the  end  of  the  tube  rests  on  one  edge  of  the  hand 
(Fig.  22),  holding  them  at  an  angle  of  about  45°.  If  held 
horizontally,  the  condensation  water,  always  present  at 
the  lower  end  of  the  agar  medium,  will  moisten  the  surface 
and  destroy  a  characteristic  growth  along  the  needle-track. 
If  held  in  a  vertical  position,  contamination  from  the  air 
cannot  be  avoided. 


FIG.  22 
Method  of  Inoculating  Media 

3.  Remove  the  cotton  stoppers  by  taking  hold  of 
the  singed  portion  only,  and  hold  also  by  the  singed  part 
only  between  the  other  fingers.     If  the  portion  of  cotton 
from  the  inside  of  the  tube  is  touched  by  the  fingers,  or 
accidentally  falls  on  the  table  or  the  floor,  it  becomes  con- 
taminated and  must  be  singed  before  replacing. 

4.  Sterilize  the  straight  platinum  needle  in  the  flame, 
holding  it  like  a  pencil.     The  platinum  wire  should  be 
heated  until  red-hot,  and  the  glass  end  passed  slowly 
through  the  flame  once  or  twice. 

5.  After  cooling   the   needle   by   plunging   into  the 
medium,  take  up  a  small  portion  of  a  colony  or  culture 
by  a  lateral  movement  on  the  end  of  the  needle,  and, 


COLLECTING   BACTERIA   FROM   THE   AIR       41 

when  removing  it  from  the  tube,  take  care  not  to  touch 
the  walls  of  the  tube.  If  any  cotton  accidentally  sticks 
to  the  mouth  of  the  tube,  it  should  be  burnt  off  with 
a  hot  platinum  needle,  and  then  the  mouth  of  the  tube 
passed  through  the  flame  before  inserting  the  needle. 

6.  Insert  the  needle  with  the  culture  into  the  sterile 
tube,  and  carefully  draw  along  the  surface  of  the  slant, 
turning    the  needle  during  the  operation  and  without 
puncturing  the  jelly. 

7.  Sterilize  the  needle  in  the  flame,  as  above,  and 
replace  the  cotton  plugs.     It  is  not  superfluous  to  repeat 
here  that  the  platinum  needle  should  always  be  flamed 
immediately  after  taking  it  up,  and  again  before  laying 
it  down. 

A  stab-culture  is  made  in  the  same  manner  as  a  slant- 
culture,  except  that  the  medium  is  punctured  centrally 
by  a  quick,  steady  movement  of  the  needle.  Care  should 
be  taken  not  to  let  the  needle  touch  the  bottom  of  the 
tube,  the  object  being  to  make  as  narrow  a  puncture  as 
possible,  and  by  touching  the  glass  the  needle  would  bend 
and  make  a  ragged  opening  in  the  medium  on  with- 
drawing. 

Inoculations  of  liquid  media  are  made  the  same  as 
agar-slants,  except  that  the  end  of  the  needle  is  gently 
rubbed  against  the  glass  below  the  surface  of  the  liquid. 
The  medium  is  then  shaken.  After  inoculation  milk- 
cultures  should  never  be  shaken  again,  as  this  might 
destroy  a  characteristic  shape  of  the  coagulum  or  break 
up  the  cream-ring. 

Inoculations  from  liquid  media  are  made  with  the 
looped  needle.  This  may  also  be  used,  if  a  considerable 
amount  of  growth  is  required  on  a  solid  medium.  The 


42       LABORATORY   GUIDE   IN   BACTERIOLOGY 

contents  of  the  loop  are  then  spread  over  the  whole  sur- 
face. 

Describe  the  appearance  of  the  colonies  as  to — 

1.  Naked-eye  appearance: 
Color. 

Shape. 

Border. 

Size  (approximately  in  millimeters). 

2.  Appearance  under  low  power  of  microscope. 

3.  Give  the  approximate  number  of  colonies  on  the 
plate. 

4.  Examine    as    to    motility    in    the    hanging-drop. 
Preparation  of  a  hanging-drop  (Fig.  23) : 


FIG.  23 

Hanging-Drop 

(After  Muir  and  Ritchie) 

1.  Clean  a  cover-slip  in  alcohol.     Pass  several  times 
through  the  flame  so  as  to  burn  the  last  traces  of  grease 
off  the  surface. 

2.  Place  a  loopful  of  pure  water  on  the  center  of  the 
cover-slip. 

3.  Flame  the  straight  platinum  needle,  and,  after 


COLLECTING    BACTERIA   FROM   THE   AIR       43 

cooling,  touch  one  of  the  colonies  and  mix  lightly  with 
the  drop  of  water  without  spreading  it.  Take  only  a 
very  minute  amount  of  culture,  so  as  to  produce  the  faint- 
est visible  cloudiness  in  the  water. 

4.  Smear  vaselin  around  the  depression  in  a  hollow- 
ground    slide,  quickly  invert    the    cover-slip    over  the 
depression,  and  gently  press  the  margin  on  the  vaselin. 

5.  Examine  in  oil,  using  very  little  light. 
Brownian  movement. — Rub  a  small  amount  of  carmine 

in  a  mortar  with  some  water  and  make  a  hanging-drop 
preparation.  When  examining  this  through  the  oil- 
immersion  lens,  it  will  be  observed  that  the  small  particles 
of  carmine  have  a  lively  vibrating  motion.  This  is  called 
" Brownian"  or  "molecular  movement,"  or  "pedesis." 
The  particles  scarcely  change  their  relative  position. 
Actively  motile  organisms,  on  the  contrary,  widely  change 
their  relative  positions.  The  movement  of  these  may  be 
slow,  snake-like,  or  like  a  fish  swimming;  or  they  may 
dart  rapidly  across  the  field. 

Now  observe  and  describe  what  changes  have  taken 
place  in  the  tube  of  broth  with  the  hair.  Compare  with 
a  sterile  tube,  noting  the  turbidity,  sediment,  odor,  etc. 
Also  examine  in  hanging-drop. 

Examine  the  tube  of  glucose-agar  containing  the  nail- 
scrapings.  Describe  the  general  appearance,  and  note 
whether  gas-bubbles  are  present. 

Finally,  from  the  stained  preparations  make  sketches 
of  the  appearance  of  the  culture  medium,  the  shape  of 
the  colonies,  and  the  morphology  of  the  organisms. 

Make  stained  preparations  of  three  different  colonies, 
using  the  three  stains;  i.  e.,  gentian- violet,  methylene- 
blue,  and  carbol-fuchsin, 


44       LABORATORY   GUIDE   IN   BACTERIOLOGY 

Method  of  making  stained  preparations — 

1.  Clean  and  flame  a  cover-slip,  or,    if    preferred, 
a  slide  may  be  used  for  this  purpose.     Cover-slips,   if 
handled  by  the  fingers,  should  be  held  by  the  edges.     Use 
as  much  as  possible  the  forceps  made  for  that  purpose. 
After  handling  the  forceps  they  should  be  sterilized  in  the 
flame. 

2.  Place  one  loopful  of  water  on  the  cover-slip. 

3.  Take  the  minutest  quantity  of  the  colony  or  cul- 
ture on  a  platinum  needle  and  mix  gently  with  water 
until  faintly  cloudy.     Burn  the  remainder  of  the  culture 
off  the  needle. 

4.  Spread  over  the  cover-slip  by  two  or  three  sweeps 
of  the  needle.     The  water  should  spread  easily  and  not 
run  together.     If  this  takes  place,  the  cover-slip  has  not 
been  cleaned  sufficiently. 

5.  Dry  by  moving  high  over  the  flame. 

6.  Pass  three  times  back  and  forth  through  the  flame 
rapidly.     This  process   precipitates  albuminous  matter 
and  causes  the  bacteria  to  adhere  firmly  to  the  glass. 

7.  Cover  with  stain  for  10-15  seconds. 

8.  Wash  in  water  and  examine.     If  satisfactory: 

9.  Float  the  cover-slip  off  the  slide  with  water.     Blot 
with  filter-paper,  dry  in  the  air  or  high  over  the  flame, 
and  mount  in  Canada  balsam. 

10.  Label  and  preserve  this  preparation. 

Try  to  avoid  the  mistake,  made  by  most  beginners, 
of  taking  too  much  growth  on  the  needle.  For  hanging- 
drop  preparations  less  material  should  be  used  than  for 
a  stained  preparation. 


CHAPTER  VII 

EXERCISES    ON    INFECTION    AND    STERILIZATION 
EXERCISE  I.      PHENOMENA  OF  INFECTION 

1.  Prepare  three  agar-plates  as  directed  in  Chap.  VI. 

2.  Touch  the  surface  of  the  jelly  in  one  plate  with 
the  tips  of  the  fingers. 

3.  Touch  the  surface  of  the  jelly  of  another  plate  with 
the  tips  of  the  fingers,  after  washing  the  hands  carefully, 

4.  Catch  a  fly  and  allow  it  to  walk  on  the  surface  of 
the  jelly  of  the  third  plate  for  a  few  seconds.     Release 
the  fly  and  replace  the  cover. 

5.  Place  these  three  plates  in  a  locker  or  thermostat 
for  24  hours.     Observe  and  describe  the  results.     Make 
hanging-drop  and  stained  preparations  of  some  of  the 
colonies  formed. 

EXERCISE  II.      PHENOMENA  OF  STERILIZATION 

1.  Make  an  infusion  of  hay  in  a  flask  with  cotton 
stopper.     (See  Appendix  I.) 

2.  Set  the  flask  aside  for  24  hours  in  a  warm  place, 
and  observe  the  results.     To  what  are  these  results  due  ? 

3.  Expose  a  tube  of  unsterilized  broth  to  steam  in 
the  "Arnold,"  another  to  steam  in  the  autoclav,  at  120° 
for  5  minutes  each. 

4.  Set  aside  in  a  thermostat,  and  observe  the  results. 

EXERCISE  III.      PHENOMENA   OF   STERILIZATION    (CON- 
TINUED) 

Action  oj  Berkejeld  and  cotton  filters. — Berkefeld  filters 
are  made  of  diatomaceous  earth,  and  are  porous  so  as 

45 


46      LABORATORY   GUIDE   IN   BACTERIOLOGY 

to  allow  liquids  to  pass  through,  but  no  solids  or  organ- 
isms. 

i.  Arrange  a  Berkefeld  filter  so  as  to  connect  with 
the  suction  pump,  and  filter  a  quantity  of  unsterilized 
broth  (Fig.  24). 


FIG.  24 
Action  of  Berkefeld  Filter 


a.  Berkefeld  filter 

b.  Filtered  liquid 

c.  Side  tube  with  cotton  filter 


d.  Intercepting  flask 

e.  Connection  with  aspirator 
/.  Rubber  hose 


2.  Set  aside,  and  observe  the  results.  The  filter  (a), 
after  being  connected  with  the  flask,  is  sterilized  in  the 
autoclav.  The  cotton  plug  at  c  prevents  the  air,  which 
is  sucked  back,  from  carrying  germs.  The  flask  d  will 


INFECTION    AND   STERILIZATION 


47 


prevent  the  water  from  running  back  and  mixing  with 
the  filtered  liquid. 

Now  arrange  a  cotton  filter  as  shown  in  Fig.  25. 
Vessel  a,  provided  with  a  rubber  stopper  (b)  with  two 
openings,  is  arranged  so  as  to  have  a  glass  tube  (c)  reach 
to  the  bottom.  This  tube  is  provided  with  cotton  at 


a.  Erlenmeyer  flask 

b.  Rubber  stopper 

c.  Glass  tube 

d.  Cotton  filter 

e.  Broth 


FIG.  25 
Action  of  Cotton  Filter 

/.  Bent   glass  tube  with   cotton  filter 

at  f. 

g.  Erlenmeyer  flask 
h.  Tube  connecting  with  aspirator 


the  top  (d)  and  some  nutritive  medium  (broth,  e)  is 
placed  inside.  Through  the  opening  a  bent  glass  tube 
leads  out,  and  this  tube  is  also  provided  with  a  cotton 
filter  at  /.  The  whole  apparatus  is  then  sterilized  in 


48       LABORATORY   GUIDE   IN   BACTERIOLOGY 

the  autoclav  at  120°  for  5  minutes,  and  connection  is 
made  through  the  flask  (g)  and  the  tube  (h)  with  the 
aspirator.  Now  aspirate  some  air  through  the  flask, 
disconnect  at  /,  and  set  aside.  Observe  the  results.  Why 
does  no  growth  take  place  ?  Remove  the  cotton  filter 
(d),  and  drop  it  into  a  flask  containing  sterile  broth; 
place  in  the  thermostat  for  18-24  hours;  note  and 
examine  the  conditions  then  present. 


CHAPTER  VIII 

STUDY  OF  YEASTS,  MOLDS,  AND  TORUL/E 
EXERCISE   I.      CULTURAL   STUDIES 

Read  carefully  the  chapter  on  yeasts  and  molds  in 
the  textbook.  Yeasts,  torulae,  and  molds  grow  better  in 
a  medium  of  a  slightly  acid  reaction  than  in  a  neutral  or 
alkaline  medium.  Wort-agar  or  gelatin  prepared  as 
described  (Appendix  I)  will  answer  very  well  for  this 
purpose. 

1.  Melt  eight  wort-agar  tubes  in  the  water-bath. 

2.  Pour  the  contents  of  two  tubes  into  sterile  Petri 
dishes,  and  set  aside  to  solidify. 

3.  Allow  the  other  six  tubes  to  solidify  so  as  to  form 
a  slanted  surface. 

4.  After  the  plates  have  cooled,  expose  them  to  the 
air  in  two  different  places  for  10  or  15  minutes. 

5.  Place  in  your  locker. 

6.  Examine  after  24  or  48  hours.     Describe  what 
you  see. 

7.  Transfer  several  mold  and  yeast  colonies  to  the 
slants  prepared. 

8.  Make   transfer   of  a   stock-culture   of  Saccharo- 
myces  cerevisiae,  or  any  other  species  of  yeast. 

NOTE. — Molds  may  easily  be  recognized  by  the  filamentous, 
cotton-like  form  of  the  colonies.  The  hyphae  extending  into  the 
air  carry  the  spores  (conidia).  By  gently  touching  these  with  a 
sterile  platinum  needle,  the  spores  may  be  transferred  to  the 
agar-slant,  and  development  will  take  place  after  24  hours. 
Colonies  of  yeasts  or  torulae  appear  smooth,  moist,  opaque, 
elevated,  and  slightly  yellowish-white,  or  sometimes  reddish. 

49 


50       LABORATORY   GUIDE   IN   BACTERIOLOGY 

These  may  be  transferred  in  the  same  manner  as  colonies  of 
bacteria.  Molds  require  very  careful  handling  for  microscopical 
demonstration.  They  are  best  examined  in  water  in  the  unstained 
condition. 

Method  of  preparing  molds  for  microscopical  exami- 
nation— 

1.  Transfer  some  of  the  growth  to  alcohol  (50  per 
cent.). 

2.  When   thoroughly   moistened,    transfer   some   of 
the  growth  to  a  drop  of  glycerin  on  a  slide. 

3.  Spread  carefully  with  a  platinum  needle. 

4.  Cover  with  a  slip  and  examine. 

5.  If  satisfactory,  the  preparation  may  be  made  per- 
manent by  painting  a  ring  of  asphalt  around  the  edge 
of  the  cover-slip. 

Molds  may  also  be  stained  in  the  following  manner: 

1.  Place  a  small  amount  of  mold  on  a  slide. 

2.  Cover  with  alcohol. 

3.  Wash  in  water. 

4.  Stain  with  gentian-violet  or  methylene-blue. 

5.  Mount  in  glycerin. 
Study  of  yeasts — 

1.  Examine  a  small  amount  of  yeast  taken  from  an 
agar-slant  in  water  under  the  high  power  of  the  micro- 
scope.    Note  the  manner  of  reproduction  by  " budding." 

2.  Prepare  a  culture  in  liquid  wort  of  Sacch.  cere- 
visiae. 

3.  Pour  the  supernatant  liquid  of  the  24-hour-old 
culture  off,  and  distribute  the  sediment  on  a  gypsum 
block. 

NOTE. — Gypsum  blocks  may  be  prepared  in  the  following 
manner:  Gypsum  (plaster  of  paris)  is  mixed  with  half  its  vol- 
ume of  water  and  quickly  placed  in  a  cylinder  of  paper.  When 


YEASTS,    MOLDS   AND    TORUL/2  51 

perfectly  dry,  the  paper  is  cut  away  and  the  block  is  placed  in  a 
suitable  vessel  (a  Stender  dish  or  a  deep,  narrow  Petri  dish  will 
answer  very  well).  The  block  and  vessel  are  then  sterilized  in 
the  hot-air  sterilizer  for  one  hour. 

4.  Pour  enough  distilled  water  around  the  gypsum 
block  to  submerge  about  one-half  of  it. 

5.  Set  aside  in  a  cool,  dark  place  for  3  or  4  days. 

6.  Examine  a  small  portion  of  the  film  on  the  surface 
of  the  gypsum  under  the  microscope  in  water. 

NOTE. — Under  very  favorable  conditions,  and  in  the  pres- 
ence of  plenty  of  oxygen,  yeasts  will  undergo  spore-formation. 
The  great  porosity  of  the  gypsum  block,  which  admits  free  com- 
munication with  the  water,  and  the  fact  that  the  surface  of  the 
block  is  exposed  to  the  air,  offer  most  favorable  conditions  for 
spore-formation,  which  takes  place  in  3  or  4  days. 

EXERCISE     II.      THE     STUDY     OF     THE     GERMINATION     OF 
SPORES 

The  germination  of  spores  may  be  studied  from  spores 
(conidia)  of  mold  fungi.  Although  somewhat  different 
from  the  germination  of  bacterial  spores,  mold  spores 
illustrate  the  general  process  very  well. 

1.  Make  a  suspension  of  spores  of  Penicillium  glau- 
cum  (common  green  mold)  in  broth. 

2.  Flame  carefully  a  hollow-ground  slide  so  as  to 
exclude  all  other  organisms,  and  paint  vaselin  around 
the  hollow  so  as  to  form  a  complete  ring. 

3.  Take  a  loopful  of  the  spore  suspension  and  place  it 
on  a  clean  flamed  cover-glass. 

4.  Invert  over  the  prepared  hollow-ground  slide,  and 
gently  press  the  edges  on  the  vaselin  so  as  to  exclude  all 
communication  with  the  air.     This  prevents  the  hang- 
ing-drop from  drying  out,  and  also  the  invasion  of  for- 
eign organisms. 


52       LABORATORY   GUIDE   IN   BACTERIOLOGY 

5.  Observe  under  the  microscope  (No.  7  objective), 
and  make  sketches  from  day  to  day  of  the  progress  in 
the  growth,  until  the  complete  organism  is  developed 
and  spores  are  again  formed. 


CHAPTER  IX 

SCHEME   FOR   ROUTINE   STUDY   OF   THE   VARIOUS 
GROUPS  OF  ORGANISMS 

This  scheme  is  to  be  followed  strictly  in  the  study  of 
all  organisms,  except  when  special  instructions  are  given, 
and  the  student  should  thoroughly  familiarize  himself 
with  the  different  steps.  The  work  of  cultivation  as 
described  in  steps  1,3,  and  5  may  be  divided  between  two 
students  working  together,  but  all  other  procedures  are 
to  be  followed  by  each  individual. 

1.  The  inoculation  of  one  slant-agar-tube  from  each 
individual  stock-culture  supplied.    These  inoculated  agar- 
tubes  are  to  be  incubated  at  37°  for  24  hours,  unless 
otherwise  instructed. 

2.  At  the  end  of  24  hours  make  from  each  agar-tube: 

a)  A  physical  description  of  the  culture  (see  Chap. 
X). 

b)  A  hanging-drop  examination. 

c)  A  Gram  stained  preparation. 

d)  An  ordinary  stained  preparation. 

Method  of  making  preparations  according  to  Gram — 

a)  Prepare  a  film  of  the  organism  to  be  examined, 
the  same  as  in  the  ordinary  stained  preparation. 

b)  Cover  with  gentian-violet  for  i  minute. 

c)  Wash  in  water,  and  remove  the  water  by  means 
of  filter-paper,  leaving  the  surface  moist. 

d)  Cover  with  Gram's  iodin  solution  for  2  minutes. 

e)  Pour   Gram's    iodin    solution   off   and,   without 
washing,  place  in  a  watch-glass,  film  side  up,  and  cover 
with  96  per  cent,  alcohol. 

53 


54       LABORATORY   GUIDE   IN   BACTERIOLOGY 

/)  Allow  to  remain  in  alcohol,  with  occasional  agita- 
tion, for  at  least  4  minutes,  or  until  no  more  stain  is 
taken  up  by  the  alcohol. 

g)  Dry  without  washing,  and  mount. 

This  stain  is  an  important  means  of  differentiating 
species  of  bacteria. 

In  order  to  be  able  to  judge  of  the  effect  of  Gram's 
iodin  solution  on  the  stain,  the  preparation  before 
mounting  may  be  washed  in  water  and  counterstained 
with  Bismarck  brown.  This  method  shows  all  foreign 
matter  brown  in  contrast  to  the  bacteria,  and  is  especially 
adapted  for  staining  of  bacteria  in  tissues,  sputum,  etc. 

It  is  a  positive  Gram's  stain  if  by  application  of  this 
method  either  the  organism  loses  none  of  the  stain,  or 
the  stain  is  dark  blue  or  dark  slate-blue.  It  is  a  negative 
stain  if  either  the  coloration  is  completely  gone,  or  only 
a  light  bluish  tinge  is  left. 

All  stained  preparations  and  all  Gram  stains  must  be 
preserved  for  inspection. 

3.  Transfer  from  agar-culture  to  the  following  media : 
Glucose-agar. 

Gelatin. 
Potato. 
Broth. 

Litmus  milk. 
Dunham's  solution. 

These  transfers,  excepting  gelatin,  are  to  be  placed 
in  the  thermostat,  unless  otherwise  ordered. 

4.  An  accurate  description  is  to  be  made  of  each 
individual  culture  (see  Chap.  X),  and  outline  sketches 
especially  of  the  gelatin  and  the  milk  cultures.      These 
descriptions  should  be  made  complete  after  24  hours, 


ROUTINE   STUDY   OF  VARIOUS   GROUPS 


55 


and  any  changes  should  be  noted  after  48  hours  and  after 
6  days.     (See  culture  charts.) 

5.   Plates  are  to  be  made  in  agar  from  a  24-48-hour- 


FIG.  26 
Thermostat  or  Incubator 

old  broth-culture  of  each  bacterium,  unless  otherwise 
directed.  These  plates  are  to  be  described  once  after 
24-48  hours.  (See  Chap.  X.) 

NOTE. — The  thermostat  or  incubator   (Fig.   26)   is  a  box 
made  of  copper  and  having  double  walls,  between  which  water 


56       LABORATORY   GUIDE   IN   BACTERIOLOGY 

circulates.  The  outer  surface  is  usually  covered  with  asbestos 
or  linoleum,  so  as  to  hold  the  heat.  The  thermostat  is  provided 
with  two  doors,  the  inner  one  of  glass  so  as  to  enable  the  observer 
to  look  inside  without  opening  it,  the  other  one  covered  like  the 
walls.  A  thermometer  reaches  through  the  upper  part,  and  sev- 
eral air-holes  are  provided  in  it,  which  permit  free  circulation 
of  air.  The  heat  is  controlled  by  Bunsen  burners  with  self- 
regulating  devices.  The  gas  supply  is  shut  off  automatically,  if 
it  should  accidentally  become  extinguished.  For  class  use  large 
incubators  are  constructed  on  the  same  principles,  with  a  number 
of  separate  compartments. 


CHAPTER  X 

METHOD  OF  DESCRIBING  CULTURES 
The  following  method  of  describing  cultures  should 
be  carefully  studied,  and  each  suggestion  should  be 
considered  in  the  description.  It  is  of  prime  importance 
that  all  cultures  in  all  media  should  be  closely  observed, 
and  accurately  described  and  sketched,  as  this  is  the 
only  method  which  furnishes  the  proper  means  of  study- 
ing and  determining  the  different  species  of  bacteria. 
By  keeping  this  scheme  in  sight  while  making  ?.  descrip- 
tion, it  will  be  possible  after  a  little  practice  to  make 
perfect  descriptions  without  its  aid. 

I.    Morphological  characters  of  bacterium. 
Size. 

Facility  and  mode  of  staining. 
Gram's  stain. 
Special  staining  qualities. 
Motility. 

Present  or  absent. 
Sluggish  or  active  movement. 
Flagella  present  or  absent. 
Capsules  present  or  absent. 
Spores  present  or  absent. 
Involution  forms. 

Make  an  accurate  sketch  of  part  of  a  field  under 
the  microscope. 
II.    Plate  cultures. 

i.    Naked-eye  appearance. 
a)   Surface  colonies. 
57 


58       LABORATORY   GUIDE   IN   BACTERIOLOGY 

Approximate  number. 

Shape:  punctiform,  lanceolate,  oval,  cir- 
cular, spindle-shaped,  conglomerate, 
irregular,  branched,  filamentous,  rosette- 
shaped. 

Color  in  transmitted  and  reflected  light. 

Approximate  diameter  in  millimeters. 

Elevation  (and  shape  of  elevation)  or 
depression. 

Translucency. 

Moist  appearance. 

Smoothness. 

Luster. 

Liquefaction  (in  gelatin  plates  only). 

Consistency:  soft,  viscid,  hard,  chalky. 
b)    Deep  colonies. 

Color. 

Shape. 

2.    Microscopic  appearance  under  low  power  (No. 
3  objective).     Care  must  be  taken  to  use  very 
little  light,  the  diaphragm  should  be  almost 
closed. 
a)    Surface-colonies. 

Shape. 

Color. 

Translucency. 

Thickness  (in  center  and  edges). 

Nucleation. 

Striation. 

Granulation  (if  present,  whether  coarse  or 
fine). 

Homogeneity. 


METHOD   OF   DESCRIBING    CULTURES  59 

Tuberculation  (tubercle-like  appearances  al 
edge). 

Edge :  entire  or  smooth,  wavy,  with  pointed 
protuberances,   serrate,   dentate,   lacer- 
ate (as  if  torn),  fringed,  hairlike  shoots, 
curled. 
b)    Deep  colonies. 

Shape. 

Color. 

Translucency. 

Granulation. 

III.  Agar-slant-culture. 

Limitation:  confinement  to  needle-track  or 
spreading.  If  spreading,  in  what  shape  ? 

Vigor:   luxuriant  or  scant. 

Color:  by  transmitted  and  reflected  light. 

Elevation  or  depression.  Elevation  more  pro- 
nounced at  edges  or  in  center. 

Translucency. 

Moistness. 

Smoothness. 

Luster. 

Coloration  of  medium. 

Pigment  production. 

Odor. 

Gas-formation:   in  culture  or  in  medium. 

IV.  Stab-culture  in  plain  agar. 

1 .  Surface-growth  (describe  like  surface-colonies.) 

2.  Stab-growth. 
Vigor. 
Extent. 
Color. 


60       LABORATORY   GUIDE   IN   BACTERIOLOGY 

Granulation. 
Outgrowths. 
Coloration  of  medium. 
Cloudiness. 
Gas-formation. 
V.    Stab-culture  in  glucose-agar. 

Describe  like  plain  agar,  and  in  addition  always 
note  presence  or  absence  of  gas-formation,  and 
presence  of  cloudiness. 
VI.    Stab-culture  in  gelatin. 

Describe  like  plain  agar,  and  in  addition  always 
note  presence  or  absence  of  liquefaction.  If 
liquefaction  is  present,  it  may  be  saucer-shaped, 
turnip-shaped,  conical,  funnel-shaped,  hori- 
zontal (extending  the  whole  diameter  of  tube), 
sack-shaped.  Cloudiness  and  presence  of 
sediment  in  liquefied  area,  and  color  and  shape 
of  sediment,  should  be  described. 
VII.  Potato- culture. 

Describe  like  agar-slant,  adding  to  it  the  eventual 

discoloration  of  the  medium. 
VIII.    Litmus  milk  culture. 

Reaction   (acid  or  alkaline,  as  indicated   by 

color). 
Coagulation :   at  ordinary  temperature  or  upon 

heating. 

Whey:   if  present,  clear  or  turbid. 
Liquefaction  of    coagulum  (proteolysis,  pep- 

tonization). 
Gas-formation. 

Decolorization  of  litmus  (complete  or  incom- 
plete). 


METHOD    OF   DESCRIBING    CULTURES  61 

Color  of  cream-ring. 
Odor. 

In  milk  the  presence  or  absence  of  coagulation  and 
proteolysis   should   always   be   noted.     Com- 
pare your  culture  always  with  a  sterile  con- 
trol milk-tube. 
IX.    Broth-culture. 

Cloudiness:    degree  and  uniformity,  scum:   ring- 

or    island-shaped. 

Precipitate,  observed  by  shaking.    Color,  forma- 
tion, diffusibility,  viscidity,  amount. 
X.    Solidified  blood-serum. 

Describe  like  agar-slant,   and  note  presence  or 

absence  of  liquefaction. 
XI.   Fermentation-tubes. 

Gas-formation  in  closed  arm,  percentage  and 
relation  of  carbon  dioxid  to  hydrogen  expressed 

TT 

by  the  formula  . 

LUp 

Growth  in  both  arms  or  in  one  arm  only,  observed 

by  cloudiness. 
Reaction:   acid  or  alkaline. 

Directions  jor  filling  out  the  first  page  of  culture- 
charts— (see  Appendix  III.) 
I.   Name  the  group  and  organism. 
II.   Read  in  the  textbook  or  references  given  so  as  to 
indicate  the  source  and  habitat. 

III.  Name  the  most  important  references,  and  read 
them. 

IV.  Morphological  characters. 

i.  Describe  the  morphology  as  observed  from 
the  stained  preparation  opposite  the  medium 
from  which  obtained. 


62       LABORATORY  GUIDE   IN   BACTERIOLOGY 

2.  Size:    approximate  estimate  in  microns. 
Note  whether  large  or  small,  thick  or  slender, 
round  or  square  ends,  etc. 

3.  Arrangement  of  bacteria:    in  groups,  chains, 
bunches,   pairs    (diplococcus) ,    sarcina   form, 
threads,  branching,  etc.     Also  note  different 
arrangements,  if  observed,  in  different  media. 

4.  Staining  powers:    It  is  sufficient  to  mark   + 
for  positive,   -  -  for  negative  stains.     Special 
stains  must  be  described  more  fully. 

5.  Motility:    If  absent,  mark  — ;   if  present,  +. 
In  the  latter  case  describe  the  character  of 
the  movement. 

6.  Spores:    Absence  noted  by   — ;    presence,  by 
+ .     This  information  is  to  be  gathered  from 
the     textbook,     unless     specially     instructed. 
If  so  mention  the  method  of  spore-stain  applied. 

7.  Note  any  peculiar  appearance  in  the  micro- 
scopical picture,   especially  involution   forms 
and  the  presence  or  absence  of  capsules.     If 
capsules  are    present,    note    the    method    of 
demonstration. 

V.   Physiological  characters. 

1.  Relation  to  temperature :  What  is  the  optimum 
temperature?     (See   textbook  or  references.) 

2.  Relation  to  free  oxygen:    aerobe,   anaerobe, 
facultative  aerobe  or  anaerobe. 

3.  Relation   to   disinfectants,    light,   desiccation, 
heat   (thermal  death-point).     (Refer  to  text- 
book or  references.) 

4.  Pigment-production:   If  present,  +;  if  absent, 
— .     In  the  former  case,  note  the  color,  dif- 


METHOD    OF  DESCRIBING    CULTURES  63 

fusibility,    solubility,    influence   of   acids   and 
alkalis. 

5.  Gas-production  in  glucose  media:  To  be  filled 
out  only  in  case  of  actual  observation.     In 
fermentation-tubes    note  the  growth  in  either 
arm  or  both  arms,  recognized  by  turbidity. 
Note  the  total  percentage  of  the  gas  formed  in 
24   and  48  hours.     Reaction  may  be  tested  by 
the  addition  of  litmus-solution.     Gas  formula 

expressed :  . 

CO  2 

6.  Acid  or  alkali  production  in  litmus  milk. 

7.  The  production  of  indol  or   nitrites,  or  both, 
is  tested  on  the  sixth  day  of  observation  in  a 
culture    in    Dunham's    peptone-solution    or 
sugar-free  broth. 

NOTE. — Indol  is  a  decomposition  product  of 
proteids  and  belongs  to  the  aromatic  series. 
Nitrites  are  the  result  of  reduction  from  nitrates. 
The  ability  of  organisms  to  produce  these  reactions 
are  of  great  importance  in  their  differentiation.  A 
control  test  with  a  tube  of  sterile  medium  should 
always  be  made. 

8.  Enzym-production :      Froteolytic    enzym-pro- 
duction  noted  by  the  liquefaction  of  gelatin  or 
casein.    Coagulative,  by  precipitation  of  casein, 
if  acid-formation  is  absent,  or  present  only  in 
quantities  less  than  0.3  per  cent.     Diastatic,  by 
the  digestion  of  starch  (potato). 

9.  Characteristic  odor. 

10.    Pathogenesis :   What  pathogenic  effect  has  the 
organism  on  man  ?     What  effect  on  animals, 


64       LABORATORY   GUIDE   IN   BACTERIOLOGY 

and    which    animals  ?    What    diseases    are 
caused  by  the  organism  in  man  or  animal  ? 

NOTE. — It  may  be  well  here  to  call  attention  to  the  terms 
"proteolysis,"  "enzym -production,"  and  "  coagulation." 

"  Proteolysis "  is  the  breaking  up  of  complex  nitrogenous 
compounds  (proteids),  rendering  them  soluble.  This  process  is 
also  expressed  by  the  terms  " peptonization "  and  "liquefaction." 
The  liquefaction  of  gelatin  is  one  kind  of  proteolysis.  Gelatin 
is  composed  of  nitrogenous  matter  (albuminoid  or  gelatinoid), 
and  it  is  for  this  reason  mainly  that  gelatin  stab-cultures  are 
made.  If  the  gelatin  is  liquefied,  we  conclude  that  the  organism 
is  capable  of  producing  a  "  proteoly tic "  enzym.  In  milk  the 
process  is  more  complex,  and  this  medium,  on  account  of  its 
composition  (fat,  milk-sugar,  casein,  lactalbumin),  offers  excel- 
lent opportunities  for  the  organism  to  develop  different  charac- 
teristics. Milk  is  one  of  the  most  important  media.  The  casein- 
ogen,  contained  in  milk  in  colloid  solution,  may  be  precipitated 
by  an  enzym  or  by  an  acid.  This  precipitate  forms  the  coagu- 
lum.  At  least  0.4  per  cent,  of  acid,  which  is  largely  lactic  acid 
produced  by  splitting  of  milk-sugar  (lactose),  is  required  for 
precipitation,  and  this  amount  of  acid  will  turn  the  blue  litmus  to 
a  decided  pink.  A  coagulum  may  also  be  produced  by  the  presence 
of  a  " coagulative "  or  "rennet "-like  enzym,  which  is  the  result 
of  the  metabolic  activity  of  the  organism.  Such  coagulation  may 
take  place  in  milk  of  an  amphoteric  or  alkaline  reaction,  as  well 
as  in  milk  of  a  slightly  acid  reaction.  The  coagulum  formed  by 
any  of  the  mentioned  agents  may  gradually  contract,  and  a  straw- 
yellow,  opalescent  liquid  is  squeezed  out,  called  "whey."  If, 
now,  the  organism  also  produces  a  proteoly  tic  enzym,  this  will 
attack  the  coagulum  and  gradually  dissolve  it  (proteolysis,  pep- 
tonization). At  first  the  coagulum  shows  a  broken-up  surface; 
lumps  separate  and  settle  to  the  bottom,  and  finally  the  coagulum 
may  completely  disappear.  Theoretically,  coagulation  is  always 
necessary  before  proteolysis  sets  in,  but  in  the  case  of  some  organ- 
isms the  proteolytic  enzym  is  so  powerful  as  to  produce  immediate 
dissolution  of  the  casein. 

Another  phenomenon  frequently  observed  in  litmus  milk  is 


METHOD   OF  DESCRIBING    CULTURES  65 

the  discolorization  of  the  litmus,  whether  this  be  pink  or  blue. 
This  is  due  to  the  fact  that  the  organism  takes  up  the  oxygen 
necessary  to  maintain  the  coloration.  It  may  be  frequently 
observed  that  at  the  surface,  where  atmospheric  oxygen  has 
access,  the  color  remains  or  is  restored.  The  color  may  also  be 
restored  by  shaking  the  milk  vigorously,  thus  bringing  it  into 
intimate  touch  with  the  oxygen  of  the  air. 

The  production  of  a  diastatic  enzym  (diastase,  amylase)  is 
demonstrated  by  gas-production  on  potatoes.  This  medium  con- 
tains starch  in  large  amounts.  The  starch  is  inverted  into  glu- 
cose by  diastase  (amylase),  and  this  is  then  fermented  with 
gas-production. 

Directions  for  filling  out  second  page  of  culture-charts — 
i .   Note  the  reaction  of  the  medium  by  the  sign  -f-  for 
acid  and  —  for  alkali  reaction. 

2.  The    incubation    temperature    may    be    (a)    37° 
(thermostat),  (b)  room  temperature,  (c)  ice-chest. 

3.  Plates  are  to  be  described  only  once  after  24  or 
48  hours,  according  to  growth.     Make  notes  in  the  first 
column  for  gelatin;  in  the  second,  for  agar-plate,  writing 
across  the  full  width  of  the  page.     Make  sketches  in  the 
special  column  reserved  for  this  purpose. 

4.  The  growth  on  the  media  3,  4,  5,  6,  7,  and  8 
(enter    here:     " glucose-agar "     only    unless    otherwise 
instructed)  to  be  made  fully  according  to  the  outline  in 
the  spaces  under  24  hours.     In  the  spaces  under  48  hours 
and  6  days  note  only  the  changes  from  the  first  day. 

5.  Make  sketches  frequently  and   accurately,  espe- 
cially from  milk  and  gelatin  media,  and  any  other  remark- 
able growth,  in  the  column  reserved  for  this  purpose; 
also  a  sketch  of  each  organism  from  a  part  of  a  field 
under  the  microscope. 

It  is  well  to  understand  that  these  directions  for  fill- 


66       LABORATORY   GUIDE   IN    BACTERIOLOGY 


3 


•  V 


l\ 


FIG.  27 
Streak-Cultures 


FIG.  28 
Stab-Cultures 


METHOD    OF  DESCRIBING    CULTURES  67 


L 


FIG.  20 
Liquefaction  of  Gelatio 


123 

FIG.  30 

1.  Gas-bubbles  in  glucose-agar  3.  Coagulation  and  peptonization   of  milk 

2.  Coagulation  of  milk  4.  Complete  peptonization  of  milk 


68      LABORATORY  GUIDE  IN   BACTERIOLOGY 

ing  out  culture-charts  are  applicable  in  the  described 
manner  to  studies  of  cultures  furnished  by  the  laboratory. 
For  determining  any  species  of  unknown  bacteria,  original 
researches  must  be  made  to  cover  all  points,  necessarily 
without  the  possibility  of  gathering  this  information  from 
textbooks  or  references.  On  the  accuracy  of  observation 
and  description  depends  the  success  of  bacteriological 
work  and  species-determination.  It  is  frequently  neces- 
sary to  employ  special  media,  or  inoculation  of  animals, 
or  such  reactions  as  the  agglutination  test,  to  determine 
properly  what  species  one  is  dealing  with. 

The  student  will  do  well  to  familiarize  himself  care- 
fully with  all  directions  and  explanations  given  in  this 
and  the  previous  chapters.  Find  a  characteristic  for 
each  item  mentioned;  otherwise  the  descriptions  will 
be  incomplete;  and  follow  the  instructions  for  routine 
work  with  all  possible  accuracy. 


CHAPTER  XI 

STUDY  OF  CERTAIN  CHROMOGENIC  BACTERIA 
EXERCISE   I.      CULTURAL   STUDIES 

1.  Read  carefully  the  following  references  to  chromo- 
genic  organisms,  and  the  production  and  chemistry  of 
pigments: 

Sternberg,  Manual  of  Bacteriology. 
Lehmann  and  Neumann. 

Members  of  this  group  are  widely  disseminated  in  the 
air,  water,  etc.     A  few  representatives  will  be  studied. 

2.  Inoculate  agar-slants  from  stock-cultures  of  Bacil- 
lus prodigiosus,  B.  pyocyaneus,  B.  violaceus,  and  Sarcina 
luiea.     Inoculate  three  slants  each  of  B.  prodigiosus  and 
B.  pyocyaneus,  and  one  each  of  Sar.  lutea  and  B.  violaceus. 

3.  Label  each  tube  with  the  name  of  the  culture 
inoculated,  the  date  of  the  stock-culture  and  the  date  of 
inoculation.     Place  the  label  on  the  side  of  the  tube 
where  the  slanted  surface  of  the  medium  is.     It  is  then 
possible  to  study  the  growth  and  see  the  label  at  the  same 
time.     Glass  pencils  for  marking  are  very  convenient. 

4.  Place  one  culture  of  each  organism  in  the  thermo- 
stat, one  culture  of  B.  prodigiosus  and  B.  pyocyaneus  in 
the  locker,  and  leave  the  others  exposed  to  sunlight. 

5.  After  24  hours  compare  the  growths  of  B.  prodigio- 
sus and  B.  pyocyaneus,  under  the  various  conditions,  in 
respect  to— 

a)  Relative  amount  of  growth. 

b)  Relative  amount  of  pigment  produced. 

What  conclusions  can  be  drawn  from  this  experiment  ? 
69 


70      LABORATORY  GUIDE   IN   BACTERIOLOGY 

6.  Note  the  characteristics  of  the  pigments :  Are  they 
diffused  through  the  medium,  or  are  they  confined  to 
the  growth  ? 

7.  Make  descriptions  of  agar-cultures ;  also  hanging- 
drop,  stained,  and  Gram  preparations. 

8.  Transfer  from   24-hour-old   agar-cultures  of  all 
organisms  to  all  media.     (See  routine  study,  Chap.  IX.) 
Potatoes  may  be  inoculated  with  the  looped  needle,  as 
the  surface  is  too  rough  to  allow  of  a  smooth  inoculation 
with  the  straight  needle. 

9.  After  all  cultures  have  been  in  the  thermostat  for 
24  hours,  make  all  descriptions  as  outlined  in  Chap.  X. 
(Record  after  48  hours  and  6  days  only  observations  of 
change.) 

CAUTION. — The  gelatin-cultures  are  sometimes  placed  by 
students  in  the  thermostat  along  with  others  through  oversight. 
This,  of  course,  completely  defeats  the  purpose  of  obtaining  a 
stab-growth,  as  the  gelatin  will  melt.  In  order  to  avoid  this 
mistake,  it  is  recommended  to  label  one  tin  cup  or  tumbler 
" Gelatin"  in  large  letters.  This  will  serve  as  a  constant  reminder 
that  gelatin  has  to  be  kept  at  room  temperature. 

10.  Make  plate-cultures  of  the  four  organisms. 
Method  oj  making  plates — 

a)  Melt  two  agar-tubes  for  each  organism  in  the  water- 
bath  and  cool  to  43°. 

b)  Transfer  3-5  loopfuls  (according  to  the  intensity 
of  the  growth,  to  be  judged  by  the  degree  of  cloudiness) 
of  the  broth-culture  to  a  sterile  tube  of  Dunham's  solu- 
tions.    (Dilution  i). 

c)  Shake   well,    avoiding   air-bubbles   as    much    as 
possible. 

d)  Transfer  4  or  5  loopfuls  from  this  suspension  to  a 
melted  agar-tube,  and  label  this  2.     (Dilution  2). 


CERTAIN   CHROMOGENIC   BACTERIA  71 

e)  Shake  this  carefully  by  rolling  the  tube  between 
the  palms  of  the  hand,  or  stir  with  the  platinum  needle, 
so  as  to  avoid  air-bubbles. 

/)  Transfer  4  or  5  loopfuls  of  this  agar-tube  (2)  to 
the  second  agar-tube  (3),  and  mix  as  above. 

g)  If  thought  necessary,  more  tubes  may  be  treated 
in  the  same  way,  resulting  in  still  higher  dilutions.  In  the 
meantime  the  inoculated  tubes  should  be  replaced  in 
the  water-bath,  so  as  to  keep  them  liquid. 

h)  Pour  the  contents  of  the  tubes,  one  after  the  other, 
into  sterile  Petri  dishes. 

i)  Tip  the  Petri  dishes  very  carefully,  so  as  to  dis- 
tribute the  medium  evenly  over  the  bottom. 

k)  Label  them  2nd,  3d,  dilution,  etc.,  with  the  name 
of  the  organism  and  the  date. 

/)    Set  aside  on  a  level  place  to  solidify. 

m)  When  solidified,  place  them  bottom  up  in  the 
thermostat,  in  order  to  avoid  moistening  of  the  surface 
by  the  condensation  water  dropping  down  from  the 
cover. 

NOTE. — If  the  surface  were  moistened,  the  colonies  would 
run  together  and  the  characteristic  appearance  be  destroyed.  Gela- 
tin plates,  on  the  contrary,  are  placed  cover  up.  Condensation 
water  does  not  form  on  these  plates  as  gelatin  may  be  liquefied 
by  the  organisms.  The  liquefied  part  would  then  fall  from  the 
medium  on  the  cover  and  ruin  the  plate. 

The  plates  prepared  in  the  above  manner  should  be 
studied  after  24  hours,  or,  if  not  sufficiently  developed, 
after  48  hours,  according  to  directions  in  Chap.  X. 

EXERCISE  II.      STUDY  OF  PIGMENTS 

On  the  sixth  day  take  agar-slant  or  potato-cultures 
of  the  four  chromogenic  bacteria,  and  proceed  as  follows : 


72       LABORATORY   GUIDE   IN  "BACTERIOLOGY 

Pour  96  per  cent,  alcohol  on  the  cultures  of  B.  prodigi- 
osus,  B.  violaceus,  and  Sar.  lutea.  The  pigment  should 
dissolve.  Filter  the  liquids  into  clean  test-tubes,  and, 
by  adding  a  few  drops  of  5  per  cent,  hydrochloric  acid, 
note  the  change  in  color.  Then  add  an  excess  of  a  2 
per  cent,  solution  of  sodium  hydrate,  and  note  whether 
or  not  the  color  returns  and  is  changed  again.  Then 
pour  chloroform  on  a  culture  of  B.  pyocyaneus.  This 
will  dissolve  the  bluish-green  pigment  (pyocyanin). 
Filter,  and  evaporate  on  the  water-bath.  When  almost 
dry,  place  a  small  amount  on  a  slide,  and  observe  the 
small  crystals  of  pyocyanin  under  the  microscope.  Note 
also  the  aromatic  odor  given  off  by  the  pigment  as  the 
solvent  evaporates. 


CHAPTER  XII 
THE  PYOGENIC  GROUP 

EXERCISE  I.      THE  PYOGENIC  GROUP  (SUBGROUP  A)1 
MEMBERS — 

Staphylococci,  streptococci,  Microc.  tetragenus. 

Inoculate  agar-slants  from  cultures  (furnished)  of 
Staphylococcus  pyogenes  aureus,  Staph.  pyogenes  albus, 
and  Streptococcus  pyogenes.  These  organisms  are  patho- 
genic, and  care  must  be  taken  to  observe  the  rules  of 
technique  with  the  utmost  accuracy.  Any  carelessness 
may  be  followed  by  the  gravest  consequences.  In  case 
of  accident,  such  as  the  spilling  of  a  culture  or  infecting 
the  hands,  thorough  disinfection — e.  g.,  with  a  solution 
of  mercuric  chlorid  (i :  1000) — is  absolutely  necessary. 

After  24  hours'  incubation  of  the  three  agar-slants, 
proceed  with  the  other  media  as  outlined  in  the  routine 
study  (Chap.  IX). 

Special  study. — A  rabbit  will  be  inoculated  intra- 
venously with  a  broth-culture  of  Staph.  pyogenes  aureus. 
The  ear  of  the  rabbit  is  carefully  shaved,  washed  with 
mercuric  chlorid  solution,  followed  by  alcohol.  Then 
0.5  c.c.  of  a  24-hour-old  culture  in  broth  is  drawn  up 
into  a  hypodermic  syringe,  which  has  been  previously 
sterilized  by  immersion  in  boiling  water  for  10  min- 
utes. The  mode  of  holding  a  rabbit  is  as  follows: 
The  left  arm  of  an  assistant  rests  against  the  hind- 
quarters of  the  rabbit  on  the  table,  while  the  two  hands 

1  This  subdividing  of  the  pyogenic  group  is  an  arbitrary 
measure  designed  simply  to  facilitate  study,  the  commoner  pyo- 
gens  being  studied  first. 

73 


74       LABORATORY   GUIDE   IN   BACTERIOLOGY 

gently  hold  the  fore-legs.  If  the  animal  struggles,  too 
much  force  should  not  be  applied,  as  this  might  injure 
or  kill  it.  The  struggles  may  be  overcome  by  wrapping 
the  animal  in  a  towel  or  any  other  similar  piece  of  cloth. 
The  needle  is  then  inserted  into  the  lumen  of  the  lower 
vein  (ramus  lateralis  posterior  of  the  vena  auricularis 
posterior),  which  has  been  pinched  between  the  fingers, 
or  by  means  of  a  forceps,  so  as  to  arrest  the  circulation. 
No  air  should  be  injected  with  the  culture,  as  this  will 
kill  the  animal.  The  hypodermic  needle  is  then  with- 
drawn and  sterilized  in  boiling  water  for  15  minutes. 

After  the  death  of  the  rabbit,  study  the  lesions  pro- 
duced by  the  organism,  and  make  cultures  on  slant-agar, 
and  smears  from  the  heart's  blood,  spleen,  and  any  foci 
of  suppuration. 

AUTOPSY  (SEE  SKETCH,  FIG.  31) 

1.  Have  the  instruments  sterilized  in  boiling  water. 

2.  Tie  the  animal  by  the  extremities  on  a  square 
board,  with  the  abdomen  upward. 

3.  Note  the  presence  of  any  external  lesions,  such  as 
swellings,  ulcerations,  etc. 

4.  Wash  with  a  solution  of  mercuric  chlorid  (i :  1000) 
followed  by  alcohol. 

5.  Lift  the  skin  over  the  pubes  with  the  forceps,  and 
with  the  scissors  make  an  incision  along  the  median  line 
well  above  the  sternal  notch;    then  diagonal  incisions 
extending  to  the  fore-  and  hind-legs. 

6.  Cut  the  skin  away  carefully  with  a  moderately 
sharp  knife,  avoiding  opening  the  abdominal  cavity,  and 
pin  to  the  board. 

7.  Open  the  abdomen  by  a  median  incision  from  the 
pubes  to  the  sternum. 


PYOGENIC  GROUP 


75 


-STOMAC  H 
h-SPLEEN 
-KIDNEY 


INGUINAL 
GLANDS  — 


FIG.  31 
Method  of  Autopsy  of  a  Guinea-Pig  (Diagrammatic) 


76      LABORATORY   GUIDE   IN   BACTERIOLOGY 

8.  Remove  the  anterior  thoracic  wall  by  cutting  away 
the  ribs  from  below  upward  on  each  side  to  the  thoracic 
apex. 

The  viscera  are  now  thoroughly  exposed.  Cultures 
and  smears  should  be  made  from  different  parts, 
especially  from  the  heart's  blood,  peritoneal  cavity, 
spleen,  liver,  and  localized  foci  of  suppuration. 

EXERCISE  II.      THE   PYOGENIC   GROUP    (SUBGROUP  B) 

MEMBERS — 

Micrococcus  lanceolatus. 
Micrococcus  gonorrhoeae. 
Micrococcus  intracellularis  meningitidis. 
Micrococcus  zymogenes. 

Prepared  cover-slips  of  Micr.  gonorrhoeae  are  furnished. 
Stain  these  with  methylene-blue  and  Gram's  method, 
study,  and  describe  the  microscopical  appearance.  This 
organism  is  extremely  difficult  to  cultivate.  It  is  a  strict 
parasite,  and  requires  special  media,  and  a  great  amount 
of  time  and  care,  for  artificial  cultivation.  For  these 
reasons,  only  the  morphology,  as  it  appears  in  gonorrheal 
pus,  and  which  is  very  characteristic,  is  studied. 

Inoculate  agar-slants  from  stock-cultures  of  Micr. 
lanceolatus  and  Micr.  zymogenes. 

REFERENCES  (Micr.  zymogenes) — 

MacCallum  and  Hastings,  Journal  of  Experimental  Medi- 
cine, Vol.  IV  (1899),  p.  521. 

Harris  and  Longcope,  Centralblatt  fur  Bakteriologie  und 
Parasitenkunde,  Part  I,  Vol.  XXX,  No.  9  (printed  in 
English). 

1.  Routine  study. — Note  particularly  the  microscopic 
appearance  of  both  organisms  and  the  action  of  Micr. 
zymogenes  on  milk  and  gelatin. 

2.  Special  study  A. — The  staining  of  capsules  from 


PYOGENIC   GROUP  77 

a  milk-culture  of  Micr.  lanceolatus .     Two  methods  may 
be  applied  for  this  stain : 

First  method  (Friedlander's  method)  : 

a)  Prepare  a  stain  by  the  following  formula: 

Glacial  acetic  acid i  part 

Saturated  alcoholic  solution  of  gentian- 
violet 5  parts 

Distilled  water 10  parts 

b)  Prepare  a  very  thin  film  in  the  usual  manner  from 
a  24-hour-old  milk-culture,  taking  care   to  spread  very 
thinly. 

c)  Cover  with  stain  for  10-15  seconds. 

d)  Wash  in  a  solution  of  sodium  chlorid  (0.85  per 
cent.). 

e)  Examine  in  a  sodium  chlorid  solution. 

/)  If  satisfactory,  float  the  cover-slip  off  with  salt 
solution,  dry,  and  mount  in  balsam.  Under  no  conditions 
should  plain  water  come  in  contact  with  this  preparation 
at  any  stage;  otherwise  no  capsules  will  appear. 

Second  method  (Welch's  method): 

a)  Prepare  a  very  thin  film  in  the  usual  manner  from 
a  24-hour-old  milk-culture. 

b)  Cover  with  glacial  acetic  acid  for  5  seconds. 

c)  Wash  acid  off  with  carbol-fuchsin. 

d)  Wash  stain  off    with  a  0.85  per  cent,  sodium 
chlorid  solution. 

e)  Examine  in  salt  solution. 

/)   If  satisfactory,  mount  in  balsam. 

Plain  water  should  not  come  in  contact  with  this 
preparation  at  any  stage.  The  capsule  should  be  ob- 
served as  a  lightly  stained  zone  with  well-defined  outline 
around  the  deeply  stained  organism. 


78      LABORATORY  GUIDE  IN   BACTERIOLOGY 

3.   Special  study  B. — Inoculation  of  mouse  with  Micr. 
lanceolatus. 

a)  Fasten  the  mouse  in  the  holder  (Fig.  32). 

b)  Shave  a  place  on  the  back  immediately  above  the 
tail. 

c)  Wash  with  a  solution  of  mercuric  chlorid  (i :  1000), 
followed  by  alcohol. 


FIG.  32 
Mouse-Holder 

d)  Inject  0.2  c.c.  of  a  milk-culture  of  Micr.  lanceo- 
latus. 

e)  When  dead,  perform  an  autopsy,  and  study  the 
lesions  in  the  usual  manner. 

/)  Make  cultures  in  milk  and  on  slant-agar  from 
the  heart's  blood  or  the  spleen. 

g)  Make  a  capsule  stain  from  the  heart's  blood, 
spleen,  or  other  organs. 


CHAPTER  XIII 
THE  INTESTINAL  GROUP 

This  chapter  is  devoted  to  the  study  of  the  "intestinal 
group"  of  organisms.  This  collective  group  may 
conveniently  be  subdivided  into  four  subgroups: 

Subgroup  i :  the  colon  group. — This  group  includes 
different  varieties  of  Bacillus  coli,  B.  lactis  aerogenes,  and 
some  species  of  B.  acidi  lactici. 

Subgroup  2 :  the  hog-cholera,  B.  enteritidis,  or  inter- 
mediate group. — This  group  includes  B.  cholerae  suis,  B. 
paratyphosus  (several  varieties),  B.  enteritidis,  and  B. 
icteroides.  The  term  "  intermediate  "  is  assigned  to  this 
group,  because  it  resembles  the  colon  group  on  the  one 
hand,  and  the  typhoid  group  on  the  other. 

Subgroup  3:  the  typhoid-dysentery  group. — This 
group  includes  B.  typhosus,  varieties  of  B.  dysenteriae, 
and  B.  jaecalis  alcali genes. 

Subgroup  4 :  the  proteus  group. — This  group  includes 
all  varieties  of  proteus  (Bact.  termo)  and  B.  cloacae. 

EXERCISE  I.      STUDY  OF  SUBGROUP  I :    THE  COLON  GROUP 

Inoculate  agar-slants  from  stock-cultures  of  B.  coli 
(two  varieties)  and  B.  lactis  aerogenes.  Also  inoculate 
one  tube  of  broth  with  B.  coli. 

These  two  types  of  B.  coli  are  differentiated  by  their 
ability  to  ferment  carbohydrates: 

A.  Those    which    ferment    dextrose,    lactose,    and 
saccharose. 

B.  Those  which  ferment  dextrose  and  lactose,  but 
not  saccharose. 

79 


8o       LABORATORY    GUIDE   IN   BACTERIOLOGY 

REFERENCES — 

Theobald  Smith,  The  Wilder  Quarter  Century   Book,  1893, 
p.  187. 

Jordan,  Journal  o/  Hygiene,  Vol.  I  (1901),  p.  295. 

Durham,  Journal  oj  Experimental  Medicine,  Vol.  V,  p.  353. 

1.  Routine  study. — Observe  carefully  the  growth  on 
potato  of  B.  lactis  aero  genes.     What  is  the  cause  of  gas- 
formation  in  this  case  ? 

2.  Special  study  A. — In  order  accurately  to  test  the 
action  of  micro-organisms  on  various  carbohydrates,  it 
is  necessary  to  eliminate  the  small  amount  of  sugar  in 
ordinary  broth  introduced  into  it  by  meat-extract,  which 
generally    contains    muscle-sugar    (glycogen).     This    is 
accomplished  by  adding  to  freshly  prepared  broth  a 
culture  of  B.  coli,  which  completely  decomposes  many 
carbohydrates,  including  muscle-sugar.     By  this  method 
a  sugar-free  broth  is  prepared,  which  may  be  used  as  a 
solvent  for  any  sugar  desired. 

Preparation  of  sugar-free  broth  for  the  fermentation- 
tube: 

1 .  Dissolve — 

In  water 400  c.c. 

Extract  of  beef i  g. 

Peptone 4  g. 

by  heat.     Broth  made  from  chopped  beef  (500  g.  to  i 
liter)  may  also  be  used  for  this  purpose. 

2.  After  cooling,  inoculate  with  a  broth-culture  of 
B.  coli  prepared  24  hours  previously. 

3.  Set  aside  in  the  locker,  or,  better,  in  the  thermostat, 
for  18-24  hours. 

4.  Boil  5  minutes  (to  kill  B.  coli),  and  filter  repeat- 
edly through  the  same  paper  until  perfectly  clear. 

5.  Divide  into  three  equal  parts  and  dissolve  2  g. 


INTESTINAL   GROUP  81 

(ij  per  cent.)  dextrose,  lactose,  and  saccharose,  respec- 
tively, in  each  part,  and  filter  again,  if  necessary. 

6.  Fill  fermentation-tubes,  taking  care  to  label  each 
one  properly,  and  sterilize  in  Arnold  on  3  consecutive 
days  for  20  minutes. 

All  gas  must  be  carefully  tilted  out  of  the  closed  arm 
of  the  tube  while  the  fluid  is  warm.  When  sterilization 
is  completed,  inoculate  one  set  of  the  fermentation-tubes 
with  B.  coli  A,  another  set  with  B.  coli  B,  and  a  third  set 
with  B.  lactis  aero  genes.  Inoculate  with  the  straight  or 
looped  needle. 

After  24  hours,  note  the  percentage  of  gas  formed. 
This  is  done  by  means  of  Frost's  fermentation-chart,  a 
model  of  which  is  represented  by  Fig.  33.  This  chart  is 
placed  between  the  open  and  closed  arms  of  the  tube, 
resting  on  the  neck  and  moved  along  until  the  extreme 
upper  end  of  the  closed  arm  is  level  with  the  top  of  the 
chart  and  parallel  with  the  vertical  lines.  The  percent- 
age may  then  be  read  off  by  the  figures  marked  at  each 
end  of  the  chart. 

After  taking  note  of  the  percentages,  replace  the  fer- 
mentation-tubes in  the  thermostat,  and  repeat  the  meas- 
urement after  another  24  hours'  incubation.  Many 
organisms  are  able  to  obtain  the  oxygen  necessary  for 
life  by  attacking  compounds — as,  for  instance,  carbo- 
hydrates— and  decomposing  them.  In  such  cases  a  tur- 
bidity is  developed  in  the  medium.  The  closed  arm  offers 
strict  anaerobic  conditions,  all  the  atmospheric  oxygen 
having  been  driven  out  by  the  heat  of  sterilization.  The 
bulb  allows  the  organism  to  come  in  contact  with  atmos- 
pheric oxygen,  and  therefore  offers  aerobic  conditions. 

Gas-production  is  by  no  means  a  constant  phenom- 


82       LABORATORY   GUIDE   IN   BACTERIOLOGY 

enon  accompanying  a  fermentation.  Carbohydrates  are 
fermented  by  many  organisms  without  gas-formation. 
The  usual  product  in  this  case  is  an  acid,  mostly  lactic 
acid.  Such  fermentations  produce  turbidity  only,  and 
no  gas.  It  is  therefore  necessary,  in  describing  them, 
to  characterize  the  process  as  fermentation  without 
gas-formation.  '. 

Method  oj  analyzing  the  gas  produced  in  the  closed  arm. 
—The  gas  consists  approximately  of  carbon  dioxid  and 
hydrogen,  which  may  be  proved  by  the  following  method: 
Fill  the  bulb  with  a  2  per  cent,  solution  of  sodium  hydrate, 
and  close  the  mouth  with  the  thumb,  taking  care  not  to 
leave  any  air  between  the  thumb  and  the  liquid.  Now 
slowly  tilt  the  gas  back  and  forth  from  the  closed  arm 
to  the  bulb  five  or  six  times,  and  finally  allow  it  to  collect 
again  in  the  closed  arm.  The  sodium  hydrate  combines 
with  the  carbon  dioxid,  and  consequently  on  releasing  the 
thumb  the  volume  of  gas  will  become  smaller  in  propor- 
tion to  the  amount  of  carbon  dioxid  absorbed.  Now 
measure  the  percentage  again  with  the  chart,  and  ascer- 
tain the  proportion  of  gas  left  in  the  closed  arm  to  the 
original  amount. 

Example  — 

Total  percentage  of  gas  before  addition  of  NaOH.  ...   45 
Percentage  left  after  absorption  by  NaOH 30 

Difference 15 

30  per  cent,  then  represents  the  amount  of  hydrogen 
left,  and  15  per  cent,  the  amount  of  carbon  dioxid  ab- 
sorbed.    The  proportion  is  expressed  by  the  formula 
H    =3o     2 

coa    15    r 


INTESTINAL   GROUP  83 

The  fact  that  this  gas  remaining  in  the  closed  arm  is 
hydrogen  may  now  be  proved  by  tilting  it  into  the  open 
arm,  which  has  previously  been  filled  with  water  and 
closed  by  the  thumb.  By  holding  a  burning  match 
over  it  and  quickly  releasing  the  thumb,  a  slight  explo- 
sion takes  place  from  the  sudden  combination  of  the 
hydrogen  with  the  oxygen  of  the  atmosphere. 

Gas-formation  by  bacteria  does  not  necessarily 
depend  on  the  presence  of  carbohydrates.  Nitrogen  is 
often  produced  from  nitrites,  and  hydrogen  sulphid  and 
ammonia  from  proteids,  especially  during  the  process  of 
putrefaction. 

3.  Special  study  B. — Test  for  indol  and  nitrites. 
What  is  indol  ?  What  biological  activity  of  the  organism 
does  the  presence  of  indol  or  nitrites,  or  both,  indicate  ? 

a)  Test  for  nitrites:  Add  to  a  culture  in  Dunham's 
solution,  or,  better,  in  sugar-free  broth,  successively  i 
drop  of  each  of  the  following  solutions: 

(1)  Sulphanilic  acid 0.5  g. 

Acetic  acid  (25$) 150  c.c. 

(2)  Naphthylamine  chlorid o.  i  g. 

Distilled  water 20  c.c. 

Acetic  acid  (25$) 150  c.c. 

A  yellowish-red  or  rose-color  shows  the  presence  of 
nitrites. 

b)  Test  for  nitrites  and  indol  combined. 

(1)  Add  to  a  culture  in  Dunham's  solution,  or  sugar- 
free  broth,  i  or  2  drops  pure  sulphuric  acid. 

(2)  Heat  gently.     Rose-color  shows  the  presence  of 
nitrites  and  indol.     If  no  reaction  takes  place,  add — 

(3)  A  few  drops  of  a  solution  of  o.i  g.  potassium  or 
sodium    nitrite    in    1000  c.c.    water.     Rose-color    then 
indicates  the  presence  of  indol  only. 


84       LABORATORY   GUIDE   IN   BACTERIOLOGY 

Perform  these  tests  with  all  the  organisms  of  the 
intestinal  group,  and  make  control  tests  in  sterile  Dun- 
ham's solution. 

4.  Special  study  C. — Make  capsule  stain  of  B. 
lactis  acrogenes  from  24-hour-old  milk-cultures.  (For 
method  see  p.  77.) 

The  study  of  B.  coll  is  of  special  importance  in  con- 
nection with  bacteriological  analyses  of  water  (see  Chap. 
XXIV).  The  presence  of  this  organism  in  large  num- 
bers indicates  sewage  contamination,  and  consequently 
the  danger  of  an  admixture  of  pathogenic  bacteria  such 
as  B.  typhosus  and  B.  dysenteriae. 

EXERCISE  II.      STUDY  OF  SUBGROUP  II 
THE    HOG-CHOLERA,    B.     ENTERITIDIS,    OR    INTERMEDIATE    GROUP 

Use  great  care  in  handling  members  of  this  group. 

Inoculate    agar-slants    from    stock-cultures    of    B. 
cholerae  suis    (bacillus   of   hog-cholera),    B.    enteritidis 
(Gartner's  bacillus),  and  B.  paratyphosus  (two  strains). 
REFERENCES — 
B.  cholerae  suis: 

Moore,  The  Pathology  of  Infectious  Diseases  of  Animals. 

McFarland,  Textbook  of  Bacteriology. 
B.  paratyphosus: 

Buxton,  Journal  of  Medical  Research,  1902,  Vol.  VIII,  p.  201. 

Wells  and  Scott,  Journal  of  Infectious  Diseases,  1904,  No.  i. 

Gushing,  Johns  Hopkins  Hospital  Bulletin,  1900,  p.  156. 

Durham,  Journal  of  Experimental  Medicine,  Vol.  V,  p.  353. 

1.  Routine  study. — Observe  particularly  the  bluish- 
green  coloration  of  the  cream-ring  in  litmus  milk,  and 
make  test  for  indol. 

2.  Special  study  A. — Inoculate  plain  sterile  milk  with 
B.  cholerae  suis.     After  8-10  days  a  clearing  of  the  milk 


INTESTINAL  GROUP  85 

will  be  observed,  due  to  a  solvent  action  of  the  alkali 
produced  by  the  organism  upon  the  proteid  content. 

3.  Special  study  B. — Inoculate  fermentation-tubes  the 
same  as  for  B.  coli.     Measure  and  test  gas.     Compare 
the  results  with  those  obtained  in  the  study  of  the  colon 
group.     What  difference  do  you  notice  ? 

4.  Special  study  C. — Inoculation  of  a  rabbit  sub- 
cutaneously  with  B.  cholerae  suis.     Subcutaneous  inoc- 
ulations of  rabbits  are  usually  made  in  the  following 
manner:    An  assistant,  in  a  sitting  position,  places  the 
rabbit  back-down  in  his  lap.     The  head  projects  beyond 
the  knees  of  the  assistant.     The  ears  and  hind-legs  are 
grasped,  and  the  animal  is  thus  held  in  position.     The 
hair  is  then  cut  off  on  a  portion  of  the  abdomen,  and  the 
place  is  treated  with  mercuric  chlorid  and  alcohol  in  the 
usual  manner.     With  sterilized  fingers  the  skin  is  then 
pulled  up,  the  syringe  inserted,  and  the  material  injected. 

After  the  rabbit  has  died,  study  the  lesions  produced 
by  the  organism,  and  make  smears  from  the  site  of  the 
inoculation,  the  heart's  blood,  and  other  organs.  Note 
the  polar  staining,  i.  e.,  stained  portions  at  the  two  ends 
of  the  cell  and  an  unstained  area  between.  Make  cul- 
tures on  agar  from  the  heart's  blood  and  other  internal 
organs. 

EXERCISE     III.      STUDY  OF  SUBGROUP  III 
THE   TYPHOID-DYSENTERY   GROUP 

Use  great  care  in  handling  members  of  this  group. 

Inoculate  agar-slants  from  stock-cultures  of  B.  typho- 
sus,  B.  dysenteriae  (Shiga),  and  B.  jaecalis  alcaligenes. 

i.  Routine  study. — Study  carefully  the  reaction  on 
milk,  and  test  for  indol.  Preserve  glucose-agar-cultures 
for  two  weeks. 


86      LABORATORY   GUIDE   IN   BACTERIOLOGY 

2.  Special  study  A. — Inoculate  fermentation-tubes  in 
the  same  manner  as  in  the  two  preceding  groups.     Ob- 
serve the  absence  of  gas-formation  but  note  growth  in 
both  arms.     Compare  the  results  with  those  of  the  colon 
and  intermediate  groups. 

3.  Special  study  B. — The  staining  of  flagella. — Read 
carefully  in  the  textbook  the  methods  of  staining  flagella. 
To  demonstrate  the  presence  of  flagella  on  B.  typhosus, 
the  following  method  will  give  good  results  (Loeffler's 
method) : 

a)  Prepare  the  mordant.     (What  is  a  mordant  ?     See 
the  textbook.) 

Tannic  acid  (25$  aqueous  solution) 10  parts 

Ferrous  sulphate  (saturated  aqueous  solution) ...    5  parts 
Fuchsin  (saturated  alcoholic  solution) i  part 

b)  Prepare  a  number  (six)  of  cover-slips  by  carefully 
flaming  them,  and  place  them  side  by  side  on  a  piece  of 
filter-paper.     (This  paper  must  be  burned  after  using). 

c)  Place  4  or  5  loopfuls  of  water  on  a  clean  slide. 

d)  Make   a   light   suspension   in   this   water   of   B. 
typhosus  from  a  24-hour-pld  agar-culture,  taking   par- 
ticular care  to  stir  the  suspension  as  little  as  possible. 

e)  Place  a  loopful  of  water  on  each  of  the  cover-slips. 
/)  Carry  over  a  loopful  of  the  suspension  on  the  slide 

to  one  of  the  cover-slips,  from  this  to  second,  from  the 
second  to  the  third,  etc. 

g)  Spread  carefully  and  allow  to  dry  in  the  air. 

h)  Cover  with  the  mordant. 

i)  Heat  over  a  small  flame  for  ij  minutes  while 
steam  rises,  or  better  heat  for  5  minutes  on  a  water-bath. 
Replace  the  evaporated  stain,  to  prevent  its  drying  on 
the  cover-slip. 


INTESTINAL  GROUP  87 

j)  Wash  thoroughly  in  water. 

k)  Drain  the  water  off  with  blotting-paper. 

/)  Cover  with  anilin-gentian- violet  or  carbol-fuchsin. 

m)  Heat  as  before  over  a  small  flame  for  ij  minutes. 

n)  Wash  thoroughly  in  water. 

0)  Examine  in  water. 

p)  If  satisfactory,  mount  in  balsam. 

4.  Special  study  C. — A ggluiination.—  Dried-blood 
method  of  Johnston:  A  drop  of  blood  of  a  typhoid- 
fever  patient  is  obtained  by  pricking  the  lobe  of  the  ear, 
previously  carefully  cleaned  and  washed  with  alcohol. 
The  blood  is  taken  up  by  a  piece  of  sterile  non-absorbent 
paper  or  on  a  sterile  aluminum  slide.  This  is  sent  to  a 
laboratory,  where  the  blood  is  dissolved  in  physiological 
salt  solution  in  such  a  manner  as  to  obtain  an  approxi- 
mate dilution  of  1:25.  This  solution  is  then  tested  with 
a  suspension  of  typhoid  bacilli,  a  young  culture  of  which 
is  constantly  kept  on  hand  for  this  purpose. 

For  laboratory  tests  the  serum  of  an  animal  (either 
a  rabbit  or  a  guinea-pig)  which  has  been  injected  with 
cultures  of  B.  typhosus,  previously  heated  for  i  hour  at 
60°.  This  process  kills  the  organisms,  but  the  toxins 
remain  active.  The  animal  is  then  bled  in  the  following 
manner:  One  of  the  ears  is  shaved,  and  the  skin  is 
washed  with  alcohol.  A  small  vein  near  the  border  is 
opened,  and  the  blood  is  collected  in  a  sterile  glass  vessel. 
If  the  animal  does  not  bleed  freely  enough,  it  must  be 
warmed,  preferably  by  means  of  a  hot-water  bag.  The 
blood  is  placed  in  the  ice-chest,  and  the  serum  is  col- 
lected after  separation. 

The  method  of  procedure  with  a  serum  obtained  in 
the  above-described  manner  is  as  follows: 


88       LABORATORY   GUIDE   IN   BACTERIOLOGY 

a)  Small  quantities  of  the  serum  are  diluted  with 
sterile  salt  solution  (0.85  per  cent.)  so  as  to  represent 
dilutions  of  1:5,  1:25,  and  1:50. 

b)  A  suspension  of  a  24-hour-old  agar-culture  of  B. 
typhosus  in  salt  solution  is  prepared.     This  suspension 
should  be  faintly  turbid  and  uniformly  so. 

c)  Three   hanging-drop   preparations   are   made  by 
mixing  a  loopful  of  this  suspension,  with  a  loopful  of  the 
three  serum  dilutions,  respectively.     The  final  dilutions 
then  are:    1:10,  1:50,  and  1:100. 

d)  Examine   with   the  high  power   (dry   lens),   and 
observe  the  clumping  of  the  bacilli,  preceded  by  the  loss 
of  motility. 

e)  Tabulate  the  results  as  to  time  and  completeness 
of  reaction. 

/)  Make  a  control  hanging-drop  without  serum  to 
test  the  motility  and  the  absence  of  clumps. 

Blood  may  also  be  obtained  by  puncturing  the  lobe 
of  the  ear  and  collecting  it  in  a  capillary  glass  tube  with 
a  small  bulb.  By  holding  the  bulb  down,  fill  three- 
fourths  full  with  blood,  and  seal  the  ends  in  the  flame. 
In  45  minutes  the  serum  will  have  separated,  and  is 
to  be  tested  in  the  above  manner. 

The  above-described  method  of  agglutination  test  is 
known  as  the  microscopic  test.  Another  method,  in 
which  larger  amounts  of  serum  and  suspension  are 
required,  is  known  as  the  macroscopic  method.  Small 
test-tubes  are  used,  and  definite  amounts  of  bacterial  sus- 
pensions are  introduced  by  means  of  finely  graduated 
sterile  pipettes.  The  serum  is  then  added  in  varying 
amounts  so  as  to  effect  the  desired  dilutions  (see  table,  p. 
117).  The  tubes  are  then  incubated  at  37°,  usually  for 


INTESTINAL    GROUP  89 

2  hours,  or  longer  if  desired.  If  complete  agglutination 
takes  place,  the  bacteria  will  have  collected  in  clumps  at 
the  bottom,  forming  a  sediment.  The  supernatant  fluid 
is  perfectly  clear.  By  varying  amounts  of  sediment  and 
varying  degrees  of  turbidity  of  the  supernatant  fluid,  the 
degree  of  agglutination  may  be  estimated.  A  control- 
tube  of  a  bacterial  suspension  without  addition  of  serum 
serves  as  a  guide.  Controls  with  normal  serum  should 
also  be  made. 

5.   Special  study  D. — 

a)  Make   cover-slip  preparations  of  B.   dysenteriae 
from  glucose-agar-cultures  10-12  days  old.     Involution 
forms  are  then  plentiful  and  can  be  studied. 

b)  Also  make  cover-slip  preparations  of  B.  typhosus 
from   glucose-agar-cultures    10-12   days  old.      Does  it 
show  the  same  picture  ? 

EXERCISE     IV.      STUDY   OF   SUBGROUP   IV 

THE  PROTEUS  GROUP 
REFERENCES — 

Lehmann  and  Neumann. 

Jordan,  State  Board  of  Health  of  Massachusetts,  1890. 

Jordan,  Journal  of  Hygiene,  Vol.  II. 

Inoculate  agar-slants  from  stock-cultures  of  Proteus 
vulgaris,  Prot.  zenkeri,  and  Bacillus  cloacae. 

1.  Routine  study. — Observe  particularly  the  action 
on  milk  and  gelatin. 

2.  Special  study  A . — Make  plates  in  gelatin  and  agar, 
and  observe  the  colonies  after  24,  48,  and    72   hours. 
Note  the  appearance  of  the  colonies  of  Prot.  vulgaris 
and  of  Prot.  zenkeri  on  both  media. 

3.  Special  study  B. — Inoculate  fermentation-tubes  in 
the  same  manner  as  for  the  other   intestinal  groups. 


90       LABORATORY   GUIDE   IN   BACTERIOLOGY 

Determine  the  percentage  of  gas  formed  and  the  gas 
formula.  Compare  the  results  with  those  of  the  other 
intestinal  groups.  How  does  the  gas  formula  differ 
from  those  previously  examined  ? 

NOTE. — In  order  to  obtain  a  clear  picture  of  the  chief  differ- 
ential characteristics  of  the  four  intestinal  groups,  it  is  recom- 
mended to  tabulate  the  results  in  parallel  columns,  as  outlined 
below.  Express  positive  results  by  the  sign  + ;  negative,  by  — . 
Complete  agglutination  is  expressed  by  +  + ;  slight,  by  -f . 


INTESTINAL   GROUP 


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CHAPTER  XIV 

THE  CAPSULATED  GROUP 
MEMBERS — 

Varieties  of  Bacillus  mucosus  capsulatus. 

B.  lactis  aero  genes. 
REFERENCE — 

Perkins,  Journal  o)  Infectious  Diseases.  1904,  No.  2. 

Inoculate  agar-slants  from  stock-cultures  of  B.  mucosus 
capsulatus  (Friedlander's  pneumo-bacillus)  and  B.  lactis 
aero  genes.1 

1.  Routine  study. — Observe  particularly  the  viscous 
condition  of  cultures  on  solid  media,  the  consistency  of 
liquid  media,  and  the  gas-formation  on  potato. 

2.  Special  study  A. — Staining  of  capsules  from  24- 
hour-old  milk-cultures  (see  p.  77). 

3.  Special  study  B. — Intraperitoneal   inoculation  of 
rabbit  with  B.  mucosus  capsulatus. 

Method  of  intra peritoneal  inoculation. — The  rabbit  is 
held  in  the  same  manner  as  described  in  Chap.  XII, 
The  hair  is  clipped  close  over  the  left  lower  abdominal 
quadrant.  Then  (after  washing  with  mercuric  chlorid 
i :  1000  and  alcohol)  pass  the  needle  at  first  beneath  the 
skin,  then,  holding  it  at  about  a  right  angle  to  the  abdomi- 
nal surface,  carefully  press  it  through  the  abdominal  wall, 
which  is  usually  made  tense  by  the  resistance  of  the 
animal.  Successful  passage  of  the  abdominal  wall  can 
be  felt  by  the  sudden  loss  of  resistance  to  the  needle's 
pressure.  Then  make  the  injection,  and  rapidly  with- 

1  B.  lactis  aerogenes,  if  studied  in  the  colon  group,  need  not 
be  studied  again 

92 


CAPSULATED    GROUP  93 

draw  the  syringe.  If  the  contents  of  the  needle  have 
been  properly  emptied  into  the  peritoneal  cavity,  no 
swelling  takes  place,  as  is  noticed  in  subcutaneous  inocu- 
lations. 

When  the  animal  has  died,  perform  an  autopsy  and 
study  the  lesions.  Make  cultures  from  the  heart  or 
internal  organs  in  the  usual  manner,  and  make  capsule 
stains  from  the  heart's  blood. 


CHAPTER  XV 
THE  DIPHTHERIA  GROUP 
MEMBERS — 

Bacillus  diphtheriae. 
B.  pseudodiphthericus. 
B.  xerosis. 

Use  great  caution  in  handling  members  of  this  group. 
Inoculate     agar-slants     from    stock-cultures    of    B. 
diphtheriae  and  B.  pseudodiphthericus. 

1 .  Routine  study. — Stain  B.  diphtheriae  with  Loeffler's 
methylene-blue  instead  of  gentian-violet.     The  staining 
may   be   facilitated   by   the   application   of   mild   heat. 
Observe  particularly  the  peculiar  effects  of  the  staining 
process,  and  make  accurate  sketches  of  what  yon  see 
under  the  microscope. 

2.  Special  study  A. — Neisser's  method  of  staining  to 
demonstrate  the  granules  in  the  bacilli: 

a)  Prepare  two  solutions. 

SOLUTION   I 

Methylene-blue i  g. 

Dissolve  in  alcohol  96$ 20  c.c. 

Add  distilled  water. .  .    950  c.c. 

Glacial  acetic  acid 50  c.c. 

SOLUTION   II 

Bismarck  brown 2  g. 

Distilled  water 1000  c.c. 

b)  Prepare  a  film  in  the  usual  manner  from  a  culture 
of  B.  diphtheriae  on  blood-serum  (furnished). 

c)  Stain  in  Solution  I  for  5  to  6  seconds. 

d)  Wash  in  water. 

94 


DIPHTHERIA  GROUP  95 

e)  Counterstain  with  Solution  II  for  10-15  seconds. 
/)  Wash  in  water,  examine  in  water,  dry,  and  mount 
in  balsam. 

3.  Special  study  B. — Test  for  acid  formation  in  a 
culture,  one  week  old,  in  neutral  glucose-broth  by  addi- 
tion of  a  few  drops  of  litmus-solution. 

4.  Special  study  C. — Cultivation  of  B.  diphtheriae 
on  eggs.     (Method  of  Wyatt  Johnston). 

NOTE. — This  method  is  recommended  as  an  emergency  cul- 
ture test,  the  egg  taking  the  place  of  Loeffler's  blood-serum. 

a)  Sterilize  over  the  flame  a  small  empty  pot  of  meat- 
extract,  or  any  other  vessel  of  suitable  size. 

b)  Carefully  break  with  sterile  forceps  the  shell  of 
a  hard-boiled  egg  at  the  blunt  end,  taking  care  not  to 
rupture  the  membrane  lining  the  shell. 

c)  Flame  the  exposed  part,  and  carefully  free  the 
coagulated  albumen  from  any  membrane. 

d)  Inoculate  by  gently  rubbing  some  culture  or  throat 
swab  on  the  exposed  egg-albumen. 

e)  Invert  and  set  in  the  sterilized  pot. 
/)  Place  in  the  thermostat,  and 

g)  Observe    the    appearance    and    make    a    stained 
preparation  after  24  hours. 

5.  Special  study  D. — Study  of  B.  xerosis.     Obtain 
mucus  from    inner   angle    of   the    eyelids    by   stroking 
with  a  platinum  loop.      Make   two   film  preparations, 
stain  one  by  Gram's  method  and  the  other  with  methy- 
lene-blue.     Try  culture   on   slant-agar   and   if   impure, 
plate    out.     Wherein    does  B.   xerosis   differ  from  the 
other  members  of  the  group  ? 

6.  Special  study  E. — 


96      LABORATORY   GUIDE   IN   BACTERIOLOGY 
EXPERIMENT   I 

a)  Procure  a  guinea-pig  (or  rabbit),  and  clip  the  hair 
over  a  small  area  on  the  surface  of  the  abdomen. 

b)  Cut  a  small  opening  in  the  skin,  and  separate  the 
skin  from  the  muscles  below  by  pushing  in  sterile  scis- 
sors.    Expand   these   slightly  and    after   closing   again 
remove.     This  forms  a  small  pocket. 

c)  Carry  i  loopful  of  a  24-hour-old  agar-culture  into 
this  pocket. 

EXPERIMENT   II 

a)  Heat  a  24-hour-old  broth-culture  in  the  water- 
bath  for  30  minutes  at  60°. 

b)  Inject  0.25  c.c.  of  this  heated  culture  subcutane- 
ously  into  another  guinea-pig  (or  rabbit) 

c)  Observe  and  compare  in  both  animals  the  results 
by  taking  note  of  the  ante-mortem  phenomena  and  the 
lesions  post-mortem.     What  difference  is  there   in   the 
activities  of  the  two  cultures  inoculated,  and  to  what  are 
these  differences  due? 


CHAPTER  XVI 

THE  HEMORRHAGIC  SEPTICEMIA  GROUP 
MEMBERS — 

Bacillus  pestis  (bacillus  of  bubonic  plague). 
B.  cuniculicida  (bacillus  of  fowl-cholera,  bacillus  of    rabbit 
septicemia,    Bacillus    der    Rinderseuche,    Bacillus    der 
Schweineseuche,  etc.). 
REFERENCES — 

Textbook:    Moore,  The  Pathology  of  Infectious  Diseases  of 
Animals. 

Inoculate  agar-slants  from  stock-culture  of  B.  cuni- 
culicida. For  obvious  reasons,  B.  pestis  will  not  be 
studied  culturally. 

1.  Routine  study. — Stain   with  Loeffler's  methylene- 
blue  and    anilin-gentian-violet.     Observe  "polar    stain- 
ing."    What  is  "polar  staining"? 

2.  Special  study. — Inoculation  of  rabbit  subcutane- 
ously   or  by   scarification.     When   dead,   study  in   the 
usual  manner,  and  observe  particularly  the  hemorrhages 
produced    in   the    serous    membranes.     Make    cultures 
from  heart's  blood,  where  large  numbers  of  bacilli  will 
be  found.     Also  make  a  stained  preparation,  and  note 
the  typical  polar  staining.  . 


97 


CHAPTER  XVII 

THE  ANTHRAX  GROUP 

MEMBERS — 

Bacillus  anthracis. 

B.  subtilis,  including  several  varieties. 

Great  caution  is  necessary  in  handling  B.  anthracis. 
Inoculate  agar-slants  from  stock-cultures  of  B.  anthra- 
cis and  B.  subtilis. 

1.  Routine  study. — Observe  particularly  the  colonies 
formed  on  agar  and  gelatin  plates, 

2 .  Special  study  A . — Make  ' '  impression  preparation ' ' 
(Klatschprdparat)   from  a  surface  colony  on  a  gelatin- 
plate. 

Method— 

a)  Clean  and  flame  a  cover-slip  carefully. 

b)  Place  on  a  colony  of  suitable  size,  and  gently  press 
down,  taking  care  not  to  press  so  hard  as  to  disturb  the 
characteristic  shape  of  the  colony. 

c)  Lift  the  cover-slip  carefully  with  the  forceps. 

d)  Dry,  fix,  and  stain  with  methylene-blue  or  by 
Gram's  method. 

e)  Examine  under  low  and  high  power  (dry  lens), 
and  sketch  what  you  see. 

3.  Special  study  B. — Staining  of  spores.     Read  care- 
fully the  different  methods  of  staining  spores.     Why  are 
special  stains  necessary  ? 

Moeller's  method: 

a)  Prepare  several  (five  or  six)  films  in  the  usual 
manner  from  24-hour-old  agar-cultures  of  B.  anthracis 

(or  B.  subtilis). 

98 


ANTHRAX   GROUP  99 

b)  Place  in  chloroform  for  2  minutes. 

c)  After  drying  in  the  air,  cover  with  a  5  per  cent, 
solution  of  chromic  acid  for  2  minutes. 

d)  Wash  thoroughly  in  water. 

e)  Cover  with  carbol-fuchsin  and  heat  for  5  minutes 
over  the  water-bath  at  100°,  or  over  a  small  flame,  sim- 
mering gently  all  the  while. 

/)  Decolorize  with  i  per  cent,  sulphuric  acid  for  25- 
30  seconds. 

g)  Wash  thoroughly  in  water. 

h)  Mount  in  water,  and  examine  under  the  micro- 
scope to  see  if  spores  are  cherry-red  and  the  protoplasm 
colorless  or  faintly  pink. 

i)  Counterstain  with  methylene-blue  for  10-15 
seconds  without  heat. 

;)  Wash,  examine  in  water,  and  then  mount  in  balsam. 

NOTE. — The  body  of  the  cell  should  appear  blue;  the  spore, 
red. 

4.  Special  study  C. — Demonstration  of  filament- 
formation. 

a)  Spread  a  loopful  of  a  broth-culture  of  B.  anthrads 
or  B.  subtilis  on  a  clean  cover-glass. 

b)  Dry  and  fix  in  the  flame. 

c)  Cover  with  strong  acetic  acid  (80  per  cent.)  for 
5-10  seconds. 

d)  Wash  in  water. 

e)  Stain  with  gentian-violet. 

/)  Examine  in  water,  dry,  and  mount  in  balsam. 
What  is  the  object  of  applying  acetic  acid  ? 

5)  Special  study  D.— Inoculate  a  guinea-pig  subcu- 
taneously  with  0.2  c.c.  of  a  24-hour-old  broth-culture  of 
B.  anthrads,  or  insert  a  loopful  of  a  24-hour-old  agar- 


ioo     LABORATORY   GUIDE   IN   BACTERIOLOGY 

culture  in  a  "pocket"  under  the  skin.  When  the  animal 
is  dead,  perform  an  autopsy,  and  observe  particularly  the 
hemorrhagic  and  gelatinous  edema  under  the  skin;  also 
the  enlarged  spleen  and  the  hemorrhagic  adrenals. 
Make  a  stained  preparation  from  the  heart's  blood,  and 
observe  the  lack  of  spores,  and  also  the  presence  of 
capsules  and  degenerate  forms,  which  do  not  stain. 
What  prevents  the  formation  of  spores  ?  How  would 
you  distinguish  B.  anthracis  from  B.  subtilis? 


CHAPTER  XVIII 
THE  SPIRILLUM  GROUP 

MEMBERS — 

Spirillum  cholerae  asiaticae. 

Sp.  of  Finkler  and  Prior. 

Sp.  Metchnikom. 

Sp.  tyrogenum. 

And  a  number  of  spirilla  indigenous  to  water. 

Great  caution  must  be  exercised  in  manipulating  the 
spirillum  of  asiatic  cholera. 

Inoculate  agar-slants  from  stock-cultures  of  Sp. 
cholerae  asiaticae,  Sp.  oj  Finkler  and  Prior,  and  Sp. 
Metchnikovi. 

1.  Routine  study. — In   addition  to  the  usual  media, 
inoculate  an  extra  tube  of  Dunham's  peptone-solution 
from  each  organism.     Observe  carefully  from  day  to  day 
the  action  of  these  three  organisms  on  gelatin,  and  com- 
pare the  results  by  tabulation.     Observe  the  formation 
of  coccoid  involution  forms  on  agar  after  3  days.     Also 
make  plates  in  gelatin,  observe  the  colonies  from  day 
to  day,  and  compare. 

2.  Special  study  A.— Test  for  the  nitroso-indol  or 
cholera-red  reaction.     (See  test  for  indol,  p.  83.)     Make 
two  tests,  using  one  of  the  cultures  in  Dunham's  solution 
after  24  hours,  the  other  after  6  days.     Compare  the 
results  of  these  two  tests. 

3.  Special  study  B—  Stain  for  flagella  by  Loeffler's 
method  (see  p.  86). 

4.  Special  study  C. — Schottelius'  enriching  method. 


102     LABORATORY  GUIDE   IN   BACTERIOLOGY 

a)  Make  a  solution  of  i  g.  Witte's  peptone  in  100  c.c. 
of  water. 

b)  Distribute  in  three  small  Erlenmeyer  flasks,  and 
sterilize  in  autoclav. 

c)  Inoculate  one  of  these  flasks  with  Sp.  chol.  asiat. 
and  B.  chol.  suis  or  any  other  motile  bacillus. 

d)  Incubate  at  37°  for  18-24  hours.     Make  a  stained 
preparation  from  the  surface  of  the  liquid. 

e)  After  that  time,  take  one  loopful  from  the  surface, 
inoculate  the  second  flask,  and  incubate  as  before. 

/)  After  18-24  hours,  make  a  stained  preparation 
from  the  surface  of  the  second  flask,  and  examine  for 
spirilla. 

g)  Transfer  a  loopful  from  the  surface  of  the  second 
flask  to  the  third  one,  and  incubate  as  before. 

h)  After  18-24  hours,  again  stain  and  examine  for 
spirilla.  By  this  time  usually  a  film  has  formed  which 
contains  the  spirilla  in  practically  pure  culture.  How  is 
this  phenomenon  explained,  and  what  is  its  epidemiologi- 
cal  value  ? 

5.  Special  study  D. — Inoculate  a  pigeon  intramuscu- 
larly with  0.5  c.c.  of  a  broth-culture  of  Sp.  Metchnikom. 
The  breast  of  the  pigeon  is  laid  bare,  washed  with  mercuric 
chlorid  and  alcohol,  and  the  syringe  is  plunged  into  the 
muscle-fibers  and  discharged.  After  death,  note  the 
peculiar  appearance,  resembling  that  of  boiled  beef. 
Make  stained  preparations  from  blood  and  muscle-juice, 
and  examine  for  spirilla. 


CHAPTER  XIX 

THE  GROUP  OF  ACID-RESISTING  BACILLI 
MEMBERS — 

Bacillus  tuberculosis. 
B.  leprae. 
B.  smegmae. 

Moeller's  grass  bacilli,  including  a  number  of  bacilli  found 
on  grass,  dung,  in  butter,  milk,  etc. 

For  obvious  reasons,  B.  tuberculosis  will  not  be  studied 
culturally.  For  comparison,  the  culture  characteristics  of 
Moeller's  grass  bacillus  are  instructive. 

Inoculate  an  agar-slant  from  a  stock-culture  of 
Moeller's  grass  bacillus. 

1.  Routine  study. 

2.  Special  study  A . — Method  of  staining  acid-resisting 
bacilli. 

a)  Pick  out  purulent  matter  from  the  sputum  oi  a 
tuberculous  patient  and  spread  carefully  on  a  cover- 
glass. 

b)  Dry  and  fix  as  usual. 

c)  Heat  for  one  minute  on  a  water-bath  at  100°,  or 
over  a  small  flame  with  carbol-fuchsin. 

d)  Decolorize  with  acid  alcohol  (2  per  cent.  HC1  in 
80  per  cent,  alcohol)  until  the  film  in  its  thin  parts  has 
lost  almost  all  its  color. 

e)  Counterstain  with  methylene-blue  for  10  seconds 
(cold). 

/)  Examine  and  mount  in  balsam. 

NOTE. — Make  a  second  preparation,  substituting  anilin- 
gentian-violet  for  carbol-fuchsin,  and  Bismarck  brown  for  methy- 
lene-blue. 

103 


io4     LABORATORY   GUIDE   IN   BACTERIOLOGY 

3.  Special    study    B. — Observe     the    lesions    in    a 
guinea-pig  or  rabbit  dead  of  tuberculosis,  which    has 
been  inoculated  about  four  weeks  previously.     Note  the 
caseous  matter  at  the  site  of  the  inoculation,  the  enlarged 
yellowish  inguinal  and  axillary  glands,  the  small  gray 
or  yellowish  tubercles  in  the  liver,  lung,  spleen,  mesentery, 
etc.     Stain  for  bacilli  from  such  sites.     Make  cultures  on 
glycerin-agar  or  dog-serum  (see  the  textbook). 

4.  Special  study   C. — Stain  Moeller's  grass  bacillus 
from  agar-culture  by  the  same  method,   omitting  the 
counters  tain. 

5.  Special  study  D. — 

a)  Grind  in  a  mortar,  previously  sterilized,  a  small 
amount  of  a  culture  of  the  grass  bacillus,  and  mix  with 
some  sterile  milk. 

b)  Make  a  cover-slip  preparation  of  this  mixture,  and 
stain  for  tubercle  bacilli.     At  what  conclusions  as  to  the 
quality  of  the  milk  would  you  arrive,  if  you  should  find 
acid-resisting  bacilli  in  a  fresh  sample  of  milk  ? 


CHAPTER  XX 
MISCELLANEOUS  BACTERIA 
Inoculate  agar-slants  from  stock-cultures  of  Bacillus 
mallei    and    Micrococcus    melitensis.      Exercise     great 
caution  in  handling  B.  mallei. 

1.  Routine  study. — Note   especially   the   growth   of 
B.  mallei  on  potato.     Also  note  the  morphology  of  Micr. 
melitensis  and  the  staining  properties  of  B.  mallei.    Is  B. 
mallei  motile  ? 

2.  Special    study. — Intraperitoneal   injection   of   B. 
mallei  from  a  broth-culture  into  a  male  guinea-pig.     Why 
do  you  select  a  male  guinea-pig?    What  is   Straus's 
method  of  diagnosis  of  glanders? 


105 


CHAPTER  XXI 
THE  ACTINOMYCES  GROUP 
Inoculate  agar-slants  from  Actinomyces  boms  (homi- 
nis)  and  Actin.  asteroides. 

REFERENCES — 

Stokes,  American  Journal  of  the  Medical  Sciences,  Novem- 
ber, 1904. 
Wright,  Jour,  of  Med.  Research,  May,  1905. 

1.  Special  study  A. — Transfer    from  agar-slants  to 
broth    and   potato    only,    and   make   descriptions   and 
stained  preparations  as  usual. 

2.  Special  study  B. — Suspend  a  small  amount  of  the 
potato-culture    in    a    physiological    salt    solution,    and 
examine  under  the  low  power. 

3.  Special  study  C. — 

a)  Examine  a  sample  of  actinomycotic  tissue  (bovine) 
in   the   fresh   state,   for   so-called   " sulphur   granules." 
Crush  some  in  salt  solution  under  a  cover-slip  and  search 
for  "  clubs,"  using  the  low  and  high  power  dry  lenses. 

b)  Crush  others,  dry,  fix,  and  stain  by  Gram,  counter- 
stain  with  eosin  or  Bismarck  brown. 

c)  Write  a  full  description  of  the  biology  of  the  organ- 
ism, illustrated  by  drawings. 

d)  What  other  actinomycetes  have  been  described 
as   pathogenic?     Give   their   names   and   the   diseases 
which  they  produce. 


106 


CHAPTER  XXII 
THE  ANAEROBIC  GROUP  OF  BACILLI 

MEMBERS — 

Bacillus  tetani. 

B.  oedematis  maligni. 

B.  aero  genes  capsulatus. 

B.  anthracis  symptomatici. 

B.  botulinus. 

And  others. 

Study  the  different  methods  of  anaerobic  cultivation  in 
the  textbook. 

1.  Special  study  A. — Park's  method. 

a)  Boil  three  tubes  of  glucose-agar  vigorously  for  5 
minutes,  to  drive  out  the  dissolved  oxygen.     (Why  is 
the  presence  of  glucose  desirable  ?) 

b)  Cool  to  43°  and  inoculate  from  stock-culture  of 
B.  tetani,  B.  oedematis  maligni,  and  B.  aerogenes  capsu- 
latus. 

c)  Solidify  rapidly  by  immersion  in  cold  water. 

d)  Cover  the  medium  with  a  thin  layer  of  liquid  paraf- 
fin. 

e)  Place  in  thermostat. 

NOTE. — The  layer  of  parraffin  effectively  excludes  the  atmos- 
pheric oxygen,  which  would  be  inhibitory  to  the  growth  of  the 
anaerobes.  The  oxygen  necessary  for  their  multiplication  is 
derived  from  nutrient  material  of  the  medium. 

2.  Special  study  B. — Wright's  modification  of  Buch- 
ner's  method. 

a)  Liquefy,  as  before,  six  glucose-agar  tubes,  the 
plugs  of  which  have  been  replaced  by  absorbent  cotton. 

107 


io8     LABORATORY   GUIDE   IN   BACTERIOLOGY 

Cool  three  to  43°,  and  inoculate  while  fluid.     Let  the 
other  three  become  solid,  and  make  stab-cultures. 

b)  Sterilize  the  cotton  stoppers  in  a  flame,  and  with 
the  forceps,  sterilized  in  a  flame,  push  the  stoppers  into 
the  test-tubes  for  the  distance  of  about  i  inch  (2-3  cm.). 

c)  Pour  into  the  tubes  (upon  the  cotton  stoppers) 
2  c.c.  of  a  saturated  solution  of  pyrogallic  acid  in  water, 
followed  by  2  c.c.  of  a  2  per  cent,  solution  of  NaOH. 

d)  Cork  the  tubes  immediately  with  rubber  stoppers, 
and  keep  upside  down. 

e)  Incubate  at  the  required  temperature. 

Upon  what  principle  does  this  method  depend  ? 
Do  the  organisms  grow  both  aerobically  and  anaerobi- 
cally  ? 

When  you  have  obtained  a  growth,  stain  the  organ- 
isms with  anilin-gentian-violet  and  by  Gram's  method. 
Describe  and  illustrate. 

3.  Special    study    C. — Cultivation     by     Buchner's 
method,  using  fruit-jars. 

a)  Into  a  Mason  fruit-jar  of  ordinary  type  deposit 
10  g.  of  pyrogallic  acid. 

b)  Smear  vaselin  around  the  mouth  of  the  jar. 

c)  Pour  into  the  jar  100  c.c.  of  a  i  per  cent,  solution 
of  NaOH. 

d)  Then  deposit  in  the  jar  culture-tubes  previously 
inoculated. 

e)  Tightly  fasten  the  cover  of  the  jar,  and  incubate 
at  37°  for  48-72  hours. 

4.  Special  study   D. — Cultivation  in  hydrogen  gas. 

a)  Inoculate  all  media  from  stock-cultures  obtained. 

b)  Fit  up  apparatus  as  shown  in  Fig.  34. 

c)  Place  culture-tubes  in  a  Novy  jar  (Fig.  34,  a). 


ANAEROBIC   GROUP   OF   BACILLI 


109 


d)  Open  the  faucet  (b)  of  the  gas-generator  (c),  con- 
taining zinc  and  hydrochloric  acid.  The  hydrogen  gas 
generated  passes  through  two  jars,  one  of  which  con- 
tains concentrated  sulphuric  acid  (d),  the  other  a  10  per 
cent,  solution  of  sodium  hydrate  (e).  Gradually  the 
Novy  jar  is  rilled  with  hydrogen  gas,  which  can  be  tested 
by  holding  a  culture-tube  over  the  opening  (/),  and  then 
over  a  burning  match  or  gas  flame.  As  long  as  any 
detonation  takes  place  the  hydrogen  is  still  mixed  with 


-I 


FIG.  34 

Anaerobic  Cultivation  in  Hydrogen  Gas 
c.  Novy  jar  e.  Sodium  hydrate  solution 

b.  Glass  cock  /.  Opening  of  Novy  jar 

c.  Gas  generator  g.  Stopper 

d.  Sulphuric  acid 

atmospheric  oxygen.  When  finally  the  hydrogen  in  the 
Novy  jar  is  pure,  close  it  off  by  turning  the  stoppers  (g) 
and  (b),  and  place  it  in  the  incubator.  The  whole  pro- 
cess occupies  about  10  or  15  minutes. 

5.  Special  study  C. — Inoculation  of  a  rabbit  with 
B.  aerogenes  capsulatus. 

a)  Shave  the  ear  of  the  rabbit. 


no     LABORATORY   GUIDE   IN   BACTERIOLOGY 

b)  Wash  with  mercuric  chlorid  solution  and  alcohol. 

c)  Inoculate  intravenously  with  0.5  c.c.  of  a  24-hour- 
old  milk-culture. 

d)  After  the  culture  has  been  fully  distributed  in  the 
circulation,  which  takes  at  the  most  3  minutes,  kill  the 
rabbit  by  a  quick  blow  on  the  back  of  the  neck. 

e)  Put  the  rabbit  in  a  warm  place — say,  on  top  of  the 
thermostat — for  18  hours,  or  from  6  to  8  hours  inside  of 
the  thermostat. 

/)  After  this  time  has  elapsed,  perform  an  autopsy. 
Note  the  crackling,  on  pressure,  over  the  axillary  or 
inguinal  regions.  The  rabbit  is  swollen  to  a  great 
extent.  Skin  the  animal  carefully,  without  opening  the 
abdominal  cavity;  then  quickly  puncture  the  abdominal 
wall  and  bring  a  flame  to  the  opening.  Note  that  the 
escaping  gas  will  burn  with  a  blue  flame.  What  is  this 
gas  ?  Also  note  the  disorganized  condition  of  the  liver, 
spleen,  and  kidney. 

g)  Make  capsule  stains  from  the  heart's  blood  or 
organs  by  Welch's  method  (modified).  The  modification 
of  Welch's  method  is  as  follows :  Proceed  in  the  manner 
indicated  on  p.  77  and,  after  washing  the  acetic  acid  off 
with  the  stain  (carbol-fuchsin  or  gentian-violet),  dry  with 
filter  paper,  heat  the  specimen  for  5  to  10  seconds  before 
washing  off  with  the  salt  solution.  Then  proceed  as 
before. 

6.  Special  study  F. — Staining  of  spores  of  B.  tetani 
and  B.  oedematis  maligni  from  3 -day-old  glucose-agar- 
cultures  (see  p.  98). 

7.  Special  Study  G. — Inoculation  of  a  white  mouse 
or  guinea-pig  with  B.  tetani  or  its  toxin  (o.oi  c.c.)  in  the 
hind-leg  or  over  the  root  of  the  tail  (if  a  mouse).     Note 


ANAEROBIC   GROUP   OF  BACILLI  in 

daily  the  condition  of  the  animal,  and  when  dead  make 
cultures  and  cover-slip  preparations  from  the  site  of  the 
inoculation. 

8.  Special  study  H. — Inoculate  a  white  rat  with  gar- 
den earth  subcutaneously  or  in  a  pocket  above  root  of 
tail.  Note  the  condition  of  the  animal  daily.  When 
dead,  make  cultures  and  cover-slip  preparations  from  the 
site  of  the  inoculation.  Would  you  expect  to  find  bacilli 
in  the  heart's  blood  or  organs  ?  Give  reason  for  your 
answer. 


CHAPTER  XXIII 

ISOLATION   OF  UNKNOWN   BACTERIA  FROM   A 
MIXTURE 

1.  Make  hanging-drop,  stained,  and  Gram  prepara- 
tions from  the  mixture.     Note  observations  and  results. 

2.  Melt  five  or  six  agar-tubes,  and  cool  to  43°. 

3.  Transfer  5  or  6  loopfulsof  the  mixture  to  a  tube 
of  liquid  agar,  from  this  to  a  second,  and  so  on  until  all 
the  melted  tubes  are  inoculated. 

4.  Pour  into  sterile  Petri  dishes,  and  mark  them  with 
successive  numbers  and  the  date.     Place  in  the  thermo- 
stat. 

5.  After  24  hours  examine  the  colonies  under  the 
low  power,  describe  them  in  the  usual  manner,   and 
transfer   to   agar-slants   all  those  which  show  different 
appearances. 

6.  Now  proceed  with  the  usual  routine  study.     Make 
hanging-drop,  stained,  and  Gram  preparations,  transfer 
to  all  the  media,  and  describe  the  culture  characteristics 
accurately.     Make  sketches  in  the  usual  manner. 

7.  Special  tests  may  become  necessary  after  24  or 
48  hours.     Such  tests  may  consist  of— 

Capsule  stain. 

Spore  stain. 

Stain  for  acid-resisting  bacilli. 

Fermentation  tests  of  all  those  which  produce  gas 
in  glucose-agar.  Also  note  the  growth,  or  lack  of  growth, 
in  the  closed  arm  of  the  fermentation-tube. 

Test  for  acid  in  neutral  broth. 

112 


ISOLATION   OF   UNKNOWN   BACTERIA         113 

Test  for  agglutination. 

Test  for  indol. 

Anaerobic  cultivation. 

Inoculation  of  animals. 

For  final  diagnosis  consult  your  notes  and  textbook. 


CHAPTER  XXIV 
BACTERIOLOGICAL  EXAMINATION  OF  WATER,  AIR 

AND  MILK 
EXERCISE     I.      BACTERIOLOGICAL     ANALYSIS     OF     WATER 

REFERENCES — 

Horrocks,  Bacteriological  Examination  of  Water. 

Prescott  and  Winslow,  Elements  o}  Water  Bacteriology. 

Fuller  and  Johnston,  Journal  of  Experimental  Medicine,  Vol. 
IV. 

Committee  Report  o}  the  American  Public  Health  Association, 
Jour,  o}  Infectious  Diseases,  Suppl.  I,  May,  1905. 

A  bacteriological  examination  of  water  is  made  for 
the  purpose  of  determining — 

1.  Bacterial  numbers. 

2.  Bacterial  species. 

3.  Sewage  contamination. 

Collection  oj  samples. — Procure  wide-mouthed,  glass- 
stoppered  bottles,  having  a  capacity  of  at  least  100  c.c. 
After  careful  cleaning  and  drying,  wrap  them  in  lead-foil, 
and  sterilize  in  the  hot-air  oven  for  i  hour  at  160°;  then 
deposit  them  in  a  metal  or  wooden  case.  The  samples 
from  surface  waters  should  be  taken  at  least  one  foot 
below  the  surface,  to  avoid  contamination  with  organisms 
from  the  air.  If  possible,  samples  should  be  plated  on  the 
spot  or  in  the  laboratory  within  an  hour  at  the  very  latest. 
But  when  a  greater  interval  of  time  must  occur,  the 
samples  should  be  taken  to  the  laboratory  packed  in  ice, 
despite  the  probability  of  thus  destroying  a  certain  per- 
centage of  the  bacterial  flora. 

Method  oj  examination. — A  number  of  pipettes  of 
various  sizes  (i  c.c.,  2  c.c.,  5  c.c.,  and  10  c.c.)  are  plugged 

114 


EXAMINATION  OF  WATER,  AIR,  AND  MILK    115 

with  cotton  and  sterilized  in  the  hot-air  oven.  Then  a 
number  of  Erlenmeyer  flasks  are  filled  with  101  c.c.  of 
distilled  water,  and  these  are  sterilized  in  the  autoclav  at 
1 20°  for  5  minutes.  About  i  c.c.  of  water  is  lost  by  evapo- 
ration during  this  process,  so  that  the  sterile  flasks  con- 
tain 100  c.c.  each.1 

Method  oj  procedure. — 

1.  With  a  sterile  pipette  remove  i  c.c.  of  water  from 
one  of  the  sterilized  dilution  flasks. 

2.  Carry  over  to  this  flask  i  c.c.  of  the  sample  after 
a  thorough  shaking.     The  dilution  is  now  i :  100.     Mark 
with  glass  pencil. 

3.  With  a  sterile  10  c.c.  pipette  remove  10  c.c.  from 
another  dilution  flask,  and  add  to  the  remainder  10  c.c. 
of  the  first  dilution.     We  now  have  a  dilution  of  i :  1000. 
(See  dilution  table,  p.  117.)    Make  a  number  of  dilutions 
in  this  manner,  carrying  the  dilutions  higher  in  proportion 
to  the  quality  of  the  water  to  be  examined. 

4.  Melt  a  number  of  agar  and  gelatin  tubes,  corre- 
sponding to  the  number  of  dilutions  made,  and  cool  to 
43.°    Read  in  reference  book  carefully  the  chapters  on 
the  importance  of  using  both  agar  and  gelatin  media. 

5.  Mix  carefully  i  c.c.  of  each  dilution  flask  with  a 
tube  of  liquified  medium,  respectively. 

6.  Pour  into  Petri  dishes. 

7.  Incubate  the  agar-plates  at    37°,  and  keep  the 
gelatin-plates  at  room  temperature.     The  mixing  of  the 
diluted  sample  and  agar  may  also  be  effected  by  pouring 
the  sample  into  a  Petri  dish  and  mixing  it  with  the  liquid 
medium.     This  method  is  not  as  convenient,  but  slightly 

1  If  work  is  carried  on  with  dilutions  of  i :  100  only,  the  flask  may 
conveniently  be  filled  with  100  c.c.  water  instead  of  101  c.c.  It  is  then  not 
necessary  to  remove  i  c.c.  of  water,  as  only  99  c.c.  are  left  after  sterilization. 


u6     LABORATORY   GUIDE   IN   BACTERIOLOGY 

more  accurate,  as  no  loss  takes  place  by  adhesion  to  the 
tube. 

Estimation  oj  colonies. — The  colonies  are  then  counted 
after  48  hours,  by  means  of  a  colony-counter  (Fig.  35). 
Plates  should  be  counted  which  contain  no  more  than 
200-300  colonies.  If  it  is  necessary  to  count  plates  with 
a  large  number  of  colonies,  an  estimate  must  be  made 
by  counting  different  sections  of  the  plate-counter  and 
averaging  the  result  for  the  whole  plate. 

Species  determination. — If  the  different  species  of 
bacteria  are  to  be  studied,  the  colonies  must  be  examined 
by  the  naked  eye  and  the  low  power.  Then  those  which 
appear  to  be  different  are  transferred  to  slant-agar  tubes, 
and  from  these  to  the  ordinary  media. 

Sewage  contamination. — The  presence  of  Bacillus 
coli  and  streptococci  is  sufficient  indication  of  sewage 
contamination  in  water. 

Method  oj  examination  jor  B.  coli  and  streptococci. — 

1.  i  c.c.  of  the  sample,  or,  if  necessary,  of  the  diluted 
sample,  is  added  to  a  series  of  ten  fermentation-tubes, 
containing  sterile  2  per  cent,  glucose-broth. 

2.  Place  in  thermostat. 

3.  Examine  after  12-18  hours. 

4.  Examine  a  loopful  of  the  sediment  in  a  stained 
preparation. 

Example. — If  i  c.c.  of  the  sample  is  added  to  each 
fermentation-tube,  and  six  show  gas-formation,  there 
would  be  six  colon  bacilli  in  each  10  c.c.  if  undiluted 
water  is  employed.  By  this  method  fairly  accurate 
results  may  be  obtained. 

Isolation  is  accomplished  by  plating  in  an  agar 
medium  containing  2  per  cent,  lactose  and  10  per  cent, 
litmus  solution. 


EXAMINATION  OF  WATER,  AIR,  AND  MILK    117 
DILUTION   TABLES    FOR   AGGLUTINATION 


Number 

Amount  of  Serum 

Amount  of  Salt 
Solution 

Final  Dilution 

I 

i  part 

2. 

i  part  of  No   i 

9  parts 

•2. 

i  part  of  No   2 

4.  . 

i  part  of  No   ^ 

9  parts 

Amount  of  Serum  or 
Serum  Dilution 

Suspension 

Fina 

1  Dilution 

2  part  clear  serum  

1  8  parts 

10 

i     "        "        "     

19     " 

20 

5  parts  dil.,  No  

IS     " 

40 

4                   No  

16     " 

5° 

2.5  "      "     No  

17-5" 

80 

2       "         "        NO  

18     " 

IOO 

i     "      "     No  

10       " 

2OO 

4     "      "     No.  2  

y 
16     " 

CQO 

2       "         "        No.  2  

18     " 

y** 

1000 

I       "         "        No.  2  

19     " 

200O 

4                   No.  3  

16     " 

5000 

2     "      "     No.  3  

18     " 

IOOOO 

i     "      "     No.  3  

19     " 

2OOOO 

4                    No.  4  

16     " 

50OOO 

2                    No.  4  

18     " 

IOOOOO 

i     "      "      No.  4  

19     " 

200OOO 

DILUTION   TABLE   FOR   WATER    OR   MILK   ANALYSIS 


Number 

Amount  of  Dilution 

Amount  of 
Sterile  Water 

Final   Dilution 

I.    .     . 

Original 

I 

2.  . 

ic.c.  of  No.  i 

OQC.C. 

IOO 

3  

2OC.C.  of  No.  2 

yyv-.v,. 

Soc.c. 

500 

4  

IOC.C.  Of  No.  2 

9oc.c. 

IOOO 

5  

50.  c.  of  No.  2 

95c.c. 

2000 

6  

2C.C.  Of  No.  2 

98c.c. 

5000 

7  

ic.c.  of  No.  2 

99c.c. 

IOOOO 

8  

5c.c.  of  No.  4 

95c.c. 

2OOOO 

9  

2c.c.  of  No.  4 

98c.c. 

50OOO 

10  

ic.c.  of  No.  4 

99c.c. 

IOOOOO 

ii  

5c.c.  of  No.  7. 

95c.c. 

2000OO 

12  

2c.c.  of  No.  7. 

98c.c. 

5OOOOO 

*3  

ic.c.  of  No.  7 

99c.c. 

I  000000 

n8     LABORATORY   GUIDE   IN   BACTERIOLOGY 
EXERCISE    II.      BACTERIOLOGICAL    EXAMINATION    OF    AIR 

An  approximate  determination  of  the  number  of 
bacteria  in  the  air  can  be  made  by  the  following  simple 
method:  Place  a  certain  amount  (50  c.c.)  of  broth  in 
an  Erlenmeyer  flask  (Fig.  36,  a).  This  flask  is  provided 
with  a  rubber  stopper  (b),  provided  with  two  holes, 
through  which  the  two  glass  tubes  (c  with  a  wide  opening 


FIG.  36 
Apparatus  for  Determining  the  Number  of  Bacteria  in  a  Definite 


a.  Erlenmeyer  flask 

b.  Rubber  stopper 
c-d.  Glass  tubes 


Volume  of  Air 

/.  Five-liter  flask 
g-h.  Glass  tubes 
*.  Pinchcock 


and  d)  lead.  Cotton  plugs  are  then  inserted  at  c  and  d, 
and  the  apparatus  is  sterilized  in  the  autoclav.  A 
large  bottle  (/),  containing  5  liters  of  water,  is  then 
provided  with  a  rubber  stopper,  and  also  with  two  glass 
tubes  (g  and  h)\  h  is  connected  with  a  short  piece  of 
rubber  hose  and  a  pinchcock  (i).  When  the  Erlen- 


EXAMINATION  OF  WATER,  AIR,  AND  MILK    119 

meyer  flask  and  contents  are  sterilized,  the  tubed  is  con- 
nected, by  means  of  the  rubber  hose  £,  with  g,  and  the 
plug  at  c  is  removed.  By  opening  the  pinchcock  i,  5 
liters  of  air  are  aspirated  through  the  broth  in  flask  a. 
The  flask  is  then  disconnected,  and  i  c.c.  is  plated  in 
agar  and  i  c.c.  in  gelatin.  The  former  is  incubated  at  37°, 
and  the  latter  kept  at  room  temperature.  After  48  hours 
the  colonies  are  counted,  and  the  result  is  multiplied  by 
50.  This  then  represents  the  amount  of  bacteria  in  5 
liters  of  air. 

EXERCISE    III.      BACTERIOLOGICAL    STUDY    OF    MILK 

The  method  for  determining  the  number  of  bacteria 
in  milk  is  fundamentally  the  same  as  for  water,  except 
that  dilutions  must  be  carried  higher,  as  milk  generally 
contains  much  larger  numbers  of  bacteria. 

Sterilization  and  pasteurization  oj  milk. — Some  of  the 
important  germs  in  milk  are  saprophytes  (which  under 
favorable  circumstances  produce  disagreeable  odors  or 
tastes),  and  such  pathogens  as  the  bacillus  of  tuberculosis 
(which  may  be  derived  from  the  cow,  or  may  be  an  acci- 
dental contamination),  the  typhoid  bacillus,  the  bacillus 
of  "summer  complaint"  in  children  (possibly  identical 
with  the  bacillus  of  epidemic  dysentery),  the  germs  of 
cholera,  diphtheria,  and  scarlet  fever.  All  these,  except 
B.  tuberculosis,  flourish  in  milk  at  its  ordinary  tempera- 
ture. 

None  of  the  methods  employed  in  sterilizing  milk 
render  it  sterile  in  the  bacteriological  sense  of  the  word, 
but  by  means  commonly  employed  most  of  the  non- 
sporing  pathogenic  bacteria  are  destroyed,  along  with  a 
large  number  of  saprophytes,  thus  rendering  the  milk 


i2o     LABORATORY   GUIDE    IN    BACTERIOLOGY 

comparatively    safe    and    less    subject    to    the   ordinary 
fermentative  changes. 

1.  Sterilization  at  100°  for  30  minutes. — Such  milk, 
if  chilled  and  kept  at  a  low  temperature,  will  remain 
unchanged  for  more  than  a  week;   but,  by  the  heating, 
certain  alterations  have  been  produced  in  its  taste  and 
nutritive  qualities  which  render  it  supposedly  less  fit  for 
food  than  when  pasteurization  is  employed. 

2.  Pasteurizing    milk. — The    changes    occurring    in 
milk,  as  above  mentioned,  begin  at  about  82°.     Sterili- 
zation at  a  low  temperature  is  accomplished  by  raising 
the  temperature  to  only  75°  for  a  period  of  20  minutes. 
This  has  been  shown  to  be  sufficient  to  kill  the  germs  of 
typhoid,  cholera,  diphtheria,  and  pyogenic  cocci.    Spored 
organisms    are    not    killed.     As    shown    by    Theobald 
Smith,    tubercle    bacilli,    when    suspended    in    distilled 
water,  physiological  salt  solution,  broth,  and  milk,  are 
destroyed  at  60°  in  15-20  minutes;  but,  if  milk  contain- 
ing tubercle  bacilli  has  its  surface  exposed  to  the  air  when 
heated  to  60°,  the  pellicle  which  forms  on  its  surface 
may  contain  living  tubercle  bacilli  after  an  exposure  of 
60  minutes. 

Study  of  the  effect  of  the  above  two  methods  of  steri- 
lization as  compared  with  each  other  and  with  unsteri- 
lized  milk: 

1.  From  the  fresh  milk  provided  make  three  agar- 
plates,  using  i,  2,  and  3  loopfuls,  respectively. 

2.  Fill  about  10  c.c.  into  each  of  ten  sterile  culture- 
tubes,  and  keep  one  at  room  temperature  and  one  in  the 
thermostat. 

3.  Treat  four  of  these  tubes  in  the  following  manner: 
Place  water  in  a  saucepan  sufficient  to  cover  completely 


EXAMINATION  OF  WATER,  AIR,  AND  MILK     121 

the  milk  when  the  tubes  are  immersed  in  it.  Raise  the 
temperature  to  75°,  and  keep  it  there  by  regulating  the 
flame.  The  tubes  of  milk  are  then  immersed  in  the 
water,  and  kept  there  for  30  minutes,  as  it  requires  about 
10  minutes  for  the  milk  in  the  tubes  to  reach  the  tempera- 
ture of  the  water.  The  tubes  are  then  taken  out  and 
cooled  quickly  by  standing  them  in  cold  water.  Place 
one  of  the  tubes  at  incubator  and  the  other  at  room 
temperature.  Aerate  the  other  two  by  shaking  vigor- 
ously for  ij  minutes.  Keep  one  of  these  at  room  tem- 
perature, the  other  in  the  thermostat. 

4.  Place  two  more  milk-tubes  in  the  Arnold  at  100° 
for  30  minutes.     Keep  one  at  room  temperature  and  one 
in  the  thermostat. 

5.  The  remaining   two   tubes   autoclav  at  120°  for 
5  minutes,  and  place  one  in  the  thermostat  and  keep  the 
other  at  room  temperature. 

6.  Note  the  conditions  of  these  ten  tubes  after  2  or 
3  days.     Compare  the  results,  and  tabulate  them.     Note 
especially  coagulation,  time  elapsed  before  coagulation 
sets  in,  gas-formation,  condition  of  whey,  film,  and  odor. 
How  are  these  differences  explained  ? 

Plates  in  lactose-litmus-gelatin  should  be  made  from 
each  of  these  tubes,  and  the  colonies  studied  and  counted. 
Subcultures  on  agar-slants  may  also  be  made,  and  the 
usual  media  inoculated  from  these,  if  the  individual 
species  are  to  be  studied. 


CHAPTER  XXV 

INFLUENCE  OF  DISINFECTANTS  ON  THE  GROWTH 
OF  MICRO-ORGANISMS 

EXERCISE   I 

1.  Prepare   fifty-seven   Hill's   test-rods.     These   are 
prepared  in  the  following  manner:     Glass  rods  about 
two  inches  longer  than  ordinary  culture-tubes  are  marked 
with  hydrofluoric  acid  by  a  circle  exactly  one  inch  from 
the  end.     A  wad  of  cotton  is  then  wrapped  around  the 
middle  of  the  rod,  and  this  is  inserted  in  a  culture-tube. 
The  rod  is  then  pushed  down  until  it  nearly  reaches  the 
bottom.     That  part  of  the  rod  which  is  free  at  the  upper 
end  is  used  for  labeling.     The  whole  apparatus  is  then 
sterilized  in  the  dry-air  oven. 

2.  Fill  two  wide-mouthed  flasks,  one  with  100  c.c. 
of  a  5  per  cent,  solution  of  carbolic  acid,  the  other  with 
100  c.c.  of  a  i  per  cent,  solution. 

3.  Fill  two  similar  flasks,  one  with  100  c.c.  of  a  solu- 
tion of  mercuric  chlorid  i :  1000,  the  other  with  a  solution 
of  i :  10,000. 

4.  Fill  two  similar  flasks,  one  with  100  c.c.  of  a  10 
per  cent,  solution  of  formalin  (40  per  cent,  formaldehyde), 
the  other  with  a  i  per  cent,  solution. 

5.  Prepare  48-hour  broth-cultures  of  Staphylococcus 
pyogenes  aureus,  Bacillus  coli   and   B.  typhosus  from 
stock-cultures. 

6.  Dip   nineteen  of   these  rods   into   each  of   these 
cultures  respectively,  to  the  depth  of  one  inch ;  set  them 
aside  in  their  tubes  to  dry  over  night  in  the  thermostat, 
after  marking  each  tube  carefully. 


INFLUENCE   OF   DISINFECTANTS  123 

EXERCISE  II 

We  have  now  six  flasks  containing  different  solutions 
of  disinfectants. 

1.  Place  in  each  one  of  these  flasks  nine  of  the  pre- 
pared rods,  three  of  which  have  been  dipped  in  the 
Staph.   pyogenes  aureus   culture,   three  in   the   B.   coli 
culture,  and  three  in  the  B.  typhosus  culture. 

2.  Take  three  rods  (one  of  each  organism)  out  of 
each  flask  after  the  lapse  of  half  a  minute,  wash  by 
gently  pouring  sterile  physiological  salt  solution  over 
them  into  a  dish  containing  mercuric  chlorid  solution 
i :  1000,  and  place   each  rod  in  a  tube  of  sterile  broth 

3.  Repeat  the  proceedings  of  step  2  .with  a  second 
series  of  rods  after  2  minutes. 

4.  Repeat  again  after  5  minutes  with  the  remaining 
series. 

5.  Place  all  tubes   (fifty-seven)   in  the  thermostat. 
Three  of  these  tubes  have  not  been  dipped  into  any  one 
of  the  six  flasks  containing  antiseptics,  and  are  incubated 
with  the  others  as  controls. 

6.  Observe  the  results  carefully  on  each  of  the  four 
successive  days,  and  on  the  last  day  prove  the  relative 
growth  by  making  agar-plates  with  i  c.c.  of  each  culture, 
and  count  the  colonies  after  24  hours. 

7.  Tabulate  the  results,  and  state  your  conclusions. 

EXERCISE   III.      INFLUENCE   OF   SUNLIGHT 
EXPERIMENT    I 

1.  Inoculate   a   flask   containing    100  c.c.   of  sterile 
water  with  B.  coli. 

2.  After  thoroughly  shaking,  take  i  c.c.  by  means  of 
a  sterile  pipette,  and  plate  in  agar.     Place  the  plate  in 
the  thermostat. 


i24     LABORATORY   GUIDE   IN   BACTERIOLOGY 

3.  Expose  the  flask  to  sunlight  for  several  hours. 

4.  Make  another  plate  with  i  c.c.  of  the  suspension, 
and  place  in  a  thermostat. 

5.  After  48  hours  count  both  plates,  and  compare 
the  results. 

EXPERIMENT  II 

1.  Melt  a  tube  of  agar  and  cool  to  43°. 

2.  Inoculate  with  B.  coli  (or  any  other  organism). 

3.  Pour  into  a  sterile  Petri  dish. 

4.  After  solidification,  turn  bottom  side  up,  and  paste 
a  strip  of  black  paper  on  the  glass,  covering  part  of  the 
surface. 

5.  Expose  to  direct  sunlight  for  several  hours,  and 
note  the  result. 

EXERCISE   IV.      INFLUENCE   OF   MOIST    HEAT 

Read  the  methods  of  determining  the  thermal  death- 
point  of  bacteria  in  the  textbook. 

1.  Prepare  six  broth-cultures  each  of  B.  coli  and  B. 
subtilis. 

2.  Place  four  cultures  of  each  organism  in  the  water- 
bath  and  heat. 

3.  Remove  one  of  each  at  40°,  one  of  each  at  60°, 
one  of  each  at  80°,  and  keep  one  of  each  for  10  minutes  at 
100°. 

4.  Place  one  tube  of  each  organism  in  the  autoclav, 
and  heat  to  120°  for  5  minutes. 

5.  Now  place  all  twelve  tubes  in  the  thermostat, 
including  one  of  each  organism  as  a  control. 

6.  After  24  hours,  make  plates  of  each  tube  in  agar, 
and  place  them  in  the  thermostat. 

7.  After  24  hours,  count  the  colonies  and  compare 
the  results. 


APPENDIX  I 
SPECIAL  MEDIA 

STANDARD    METHOD    OF    PREPARING    BROTH,     NUTRIENT 

GELATIN   AND   NUTRIENT   AGAR 
(Report  of  American  Public  Health  Assocciation,Vo\.  XXX,  1905.) 


BROTH 

GELATIN 

AGAR 

i. 

Boil  15  g.  thread  agar  in  500 
c.c.  water  for  half  an  hour 
and  make  up  weight  to  500 
g.,  or  digest  for  10  minutes 
in  the  autoclav  at  no? 
Let  this  cool  to  about  60? 

2.  Infuse  500  g.  lean  meat  24 
hours  with  1000  c.c.  of  dis- 
tilled water  in  refrigerator. 

Ditto. 

Infuse  500  g.  lean  meat 
24  hours  with  500  c.c.  of 
distilled  water  in  refriger- 
ator. 

3.  Make  up  any  loss  by  evap- 
oration. 

Ditto. 

Ditto. 

4.  Strain     infusion     through 
cotton  flannel. 

Ditto. 

Ditto. 

5.  Weigh  filtered  infusion. 

Ditto. 

Ditto. 

6.  Add  i  %  Witte's  peptone. 

Ditto. 
And  10  %  gold  la- 
bel sheet  gelatin  . 

Add  2  %  Witte's  peptone. 

7.  Warm  on  water-bath  stir- 
ring   till    peptone   is  dis- 
solved, and  not    allowing 
the    temperature    to    rise 
above  60°. 

Warm  on  water- 
bath  until  pep- 
tone and  gela- 
tin are  dis- 
solved,  not 
above  60°. 

Warm  on  waterbath  until 
peptone  is  dissolved, 
not  above  60°. 

8. 

To  500  g.  of  meat  infusion 
add  500  c.c.  of  the  3% 
agar,  keeping  the  tem- 
perature below  60°. 

9.  Heat  over  boiling  water  (or  steam)  for  30  minutes. 
10.  Restore  loss  by  evaporation. 

u.  Titrate  after  boiling  one  minute  to  expel  carbonic  acid. 
12.  Adjust  reaction  to  +  i.o  %  by  adding  normal  hydrochloric  acid  or  sodium 
hydrate  as  required. 

125 


126    LABORATORY   GUIDE   IN   BACTERIOLOGY 

13.  Boil  2  minutes  over  free  flame,  constantly  stirring. 

14.  Make  up  loss  by  evaporation. 

15.  Filter   through   absorbent   cotton  and   cotton  flannel,  passing   the  filtrate 

through  the  filter  until  clear. 

1 6.  Titrate  and  record  final  reaction. 

17.  Tube,  using  10  c.c.  in  each  tube  in  the  case  of  gelatin  and  agar. 

1 8.  Sterilize  5  minutes  in  the  autoclav  at  120°,  or  for  30  minutes   in  streaming 

steam  on  3  successive  days.     Put  at  once  into  ice- water  till  solidified. 

19.  Store  in  the  ice-chest  in  a  moist  atmosphere  to  prevent  evaporation. 

Blood-serum — 

i.    Fresh  ox  blood  collected  in  sterile  jars  (museum  jars) 


FIG.  37 
Koch  Inspissator 

is  set  in  the  ice-chest  until  the  serum  has  separated.      The 
serum  is  filtered  if  necessary. 

2.   Take  3  parts  of  this  freshly  prepared  serum  and  i  part 
of, broth  containing  1.5  per  cent,  glucose,  and  mix. 


SPECIAL  MEDIA  127 

3.  Tube  in  the  usual  manner,  and  place  in  the  Koch 
inspissator  (Fig.  37)  two  or  three  rows  deep. 

4.  Incline  the  inspissator  to  the  proper  angle,  so  as  to 
produce  a  large  sloping  surface  of  the  serum. 

5.  See  that  the  water-jacket  contains  sufficient  water. 
Place  a  Bunsen  burner  below,  slowly  heat  the  water  to  the 
boiling-point,  and  boil  for  5  minutes.     Then  turn  out  the  gas. 

6.  Repeat  this  process  upon  the  two  following  days  at 
the  same  hour. 

NOTE. — Always  place  about  25  c.c.  of  water  in  the  inspissator 
along  with  the  tubes,  so  as  to  keep  up  a  saturation  of  the  air  in 
the  apparatus.  By  doing  this,  one  avoids  giving  the  serum  a  dry 
surface.  See  that  the  inspissator  contains  a  rack  made  of  wooden 
or  metal  strips,  which  keeps  the  tubes  from  touching  the  bottom 
and  wall ;  otherwise  the  serum  will  be  overheated  forming  bubbles 
in  the  mass. 

Litmus -solution. — Dissolve  i  part  of  Merck's  pure  extract 
of  litmus  in  100  parts  of  water,  filter  through  paper,  and  steri- 
lize. 

Glycerin-broth. — Add  6  per  cent,  of  pure  glycerin  to 
ordinary  broth. 

Glucose-gelatin. — Add  i  per  cent,  glucose  to  ordinary 
gelatin. 

Litmus-lactose-agar  (for  plating). — Add  i  per  cent,  of 
lactose  to  sugar-free  agar,  distribute  8  c.c.  in  culture-tubes,  and 
add  i  c.c.  of  sterile  litmus-solution  to  each  tube  before  using. 

Litmus -lactose- gelatin  (for  plating}. — Prepare  gelatin  in 
the  usual  manner,  using  10-12  per  cent,  of  gelatin,  and  dis- 
solve i  per  cent,  of  lactose.  Distribute  8  c.c.  into  culture- 
tubes,  and  add  litmus-solution  to  each  tube  before  using. 

NOTE — If  i  c.c.  of  the  liquid  to  be  analyzed  is  added,  14$ 
of  gelatin  should  be  used. 

Litmus -mannit-agar . — Prepare  like  litmus-lactose-agar, 
substituting  i  per  cent,  mannit  in  place  of  lactose. 

Neutral-red  agar. — Add  enough  0.5  per  cent,  neutral-red 


i28    LABORATORY   GUIDE   IN   BACTERIOLOGY 

solution  to  nutrient  agar  containing  i  per  cent,  glucose  to 
produce  a  clear  red  color  without  adjusting  reaction. 

Litmus-broth. —  Add  litmus-solution  to  ordinary  broth  in 
the  proportion  of  10  per  cent. 

Glycerin-agar . — Add  6  per  cent,  pure  glycerin  to  nutrient 
agar. 

Beerwort- gelatin — Autoclav  beerwort  at  120°  for  5  min- 
utes to  precipitate  proteids.  After  cooling,  filter,  dissolve  10 
per  cent,  gelatin,  and  clarify  with  white  of  egg. 

Beerwort-agar. — Prepared  like  beerwort-gelatin,  substi- 
tuting 1 1  per  cent,  agar  in  the  place  of  gelatin. 

Blood-agar. — A  drop  of  blood,  obtained  with  aseptic 
precautions,  is  smeared  on  the  surface  of  slant-agar. 

Litmus-whey  (Petruschk-y,  modified  by  Durham). — 
Casein  is  precipitated  from  milk  with  rennet-extract;  the 
whey  is  neutralized  with  4  per  cent,  citric-acid  solution  and 
heated  on  the  water-bath  for  half  an  hour.  It  is  then  filtered, 
and  litmus-solution  is  added  until  a  decided  blue  color  is 
obtained. 

Whey-gelatin. — Add  10  per  cent,  gelatin  to  clarified  whey. 

Alkaline  blood-serum  (Lorrain  Smith). — Add  1-1.5  P61" 
cent,  of  a  10  per  cent,  solution  of  sodium  hydrate  to  blood- 
serum,  tube,  and  sterilize  in  Arnold  or  in  Koch's  serum 
inspissator. 

Bread-paste  medium. — Bread  is  cut  into  slices,  dried  in 
the  oven,  and  pulverized;  it  is  then  distributed  in  100  c.c. 
flasks  until  the  layer  on  the  bottom  is  half  an  inch  thick. 
Water  is  gradually  run  in  to  cover  the  surface  of  the  bread 
Sterilize  in  Arnold. 

Mac  Conkey's  bile-salt-agar. — 

Nutrient  agar 100  c.c. 

Sodium  taurocholate 0.5$ 

Peptone 2.0$ 

This  is  boiled,  clarified,  and  filtered,  and  then  2  per  cent, 
lactose  is  added,  tubed,  and  sterilized  in  the  medium. 


SPECIAL   MEDIA  129 

Mac  Conkey' s  bile-salt  broth. — 

Sodium  taurocholate 0.5$ 

Peptone 2.0% 

Glucose o .  5$ 

are  dissolved  in  beef -broth  by  boiling;  then  filter  and  add 
litmus-solution. 

Parietti's  solution — 

Carbolic  acid 5  c.c. 

Hydrochloric  acid 4  c.c. 

Water 100  c.c. 

Eisner's  medium. — i  pound  of  sliced  potatoes  is  grated 
with  a  liter  of  water,  and  the  juice  expressed.  After  filtering 
for  24  hours,  add  i  per  cent,  peptone,  i  per  cent,  potassium 
iodid,  and  10  per  cent,  gelatin. 

Glycerinated  potato. — Prepare  potatoes  in  the  ordinary 
manner  and  soak  in  a  25  per  cent,  solution  of  glycerin.  Use 
a  glycerin -solution  in  the  bottom  of  the  tube. 

Hay-infusion. — 10  g.  of  chopped  hay  are  macerated  with 
1000  c.c.  of  water  in  the  water-bath  for  3  hours.  Filter  and 
sterilize  in  autoclav  for  10  minutes  at  120°. 

Wine-must. — Wine-must  is  diluted  with  four  times  its 
weight  of  water.  Dissolve  0.5  per  cent,  ammonium  tartrate, 
macerate  in  the  water-bath  for  i  hour,  filter,  and  sterilize  in 
Arnold  for  3  consecutive  days. 

Winogradsky' s  solution  (for  nitric  organisms). — 

Dissolve  in  water 1000  c.c. 

Potassium  phosphate i .  oo  g. 

Magnesium  sulphate o .  50  g. 

Calcium  chlorid o .  01  g. 

Sodium  chlorid 2 .  oo  g. 

Distribute  20  c.c.  in  flasks,  and  add  to  each  flask  a  small 
amount  of  magnesium  carbonate.  Sterilize  in  Arnold  for  3 
consecutive  days;  then  add  to  each  flask  2  c.c.  of  a  sterile 
2  per  cent,  solution  of  ammonium  sulphate. 


130     LABORATORY   GUIDE   IN   BACTERIOLOGY 

Yeast-water. — i  liter  of  washed  yeast  or  i  pound  of  pressed 
yeast  is  boiled  with  2  liters  of  water  for  i  hour.  The  reaction 
is  made  neutral  to  phenolphthalein,  and  the  solution  is  filtered 
until  clear,  and  sterilized  in  Arnold  for  3  consecutive  days. 

Dextrose-yeast-water. — 10-15  per  cent,  dextrose  is  dissolved 
in  yeast-water  without  adjusting  the  reaction. 

Egg  medium  (Dorset). — Eggs  are  broken  into  a  flask,  and 
the  yolks  are  broken  with  a  platinum  wire.  Then  the  flask 
is  gently  shaken,  so  as  to  mix  the  yolks  with  the  whites  without 
causing  foam  to  form.  10  c.c.  are  distributed  in  culture- 
tubes,  and  then  the  medium  is  hardened  in  a  sloping  position 
in  the  Koch  inspissator  for  2  successive  days  for  4  or  5  hours 
at  70°. 

Egg-yolk  medium  (Dorset). — Add  5-10  c.c.  of  sterile  water 
to  the  yolks  of  3  or  4  eggs,  and  then  pursue  the  same  course 
as  in  the  other  egg  medium. 

Egg-yolk  medium  (Capaldi). — A  few  loopfuls  of  egg-yolk 
are  added  to  a  tube  of  liquefied  agar  cooled  to  45-47°. 

Enriching  solution  for  B.  typhosus  (Hoffmann  and  Fischer). 
—To  peptone-meat-broth  add  i  per  cent,  nutrose,  0.5  per 
cent,  caffein,  and  i  per  cent,  solution  of  crystal  violet. 

Endo's  medium. — 

Nutrient  agar  (3$) 1000  c.c. 

Lactose 10  g. 

Alcoholic  solution  of  fuchsin 5  c.c. 

Sodium  sulphite  (10$  solution) 25  c.c. 

Sodium  hydrate  (10$  solution) 10  c.c. 

Drigalski  and  Conradi's  medium  (Modified  by  Harris). — 

Dextrose-free  broth 2000  c.c. 

Nutrose 20  g. 

Agar 40  g- 

Boil,  dissolve,  neutralize  to  phenolphthalein,  autoclav  at  120° 
for  5  minutes.  Clarify  with  whites  of  four  eggs  and  filter.  Then 
add— 


SPECIAL   MEDIA  131 

Lactose 30  g. 

Litmus-solution 260  c.c. 

Crystal  violet  (o.  \%  aqueous  solution) 20  c.c. 

Tube  and  sterilize  once  in  Arnold. 

Uschinsky's  non-proteid   medium   (FraenkePs    modifica- 
tion) . — 

Disodium  hydrogen  phosphate 20  g. 

Ammonium  lactate 63  g. 

Asparagin 34  g. 

Sodium  chlorid 50  g. 

Water 10,000  c.c. 

Phenol  media. — i  part  carbolic  acid  is  added  to  1000 
parts  medium. 

Dog-blood  serum. — Serum  of  dog's  blood  is  coagulated  in 
slanted  tubes  in  the  Koch  inspissator  for  3  hours  at  75°. 

Nitrate  broth. — Add  5  parts  of  potassium  nitrate  to  each 
1000  parts  of  ordinary  broth. 

Nitrate  solution. — 5  c.c.  of  2  per  cent,  aqueous  potassium- 
nitrate  solution  are  added  to  a  solution  of  i  g.  of  peptone  in 
1000  c.c.  of  water. 


APPENDIX  II 
STAINING  SOLUTIONS 
Delafield's  hematoxylin — 

Hematoxylin  crystals 4  g. 

Alcohol .25  c.c. 

Ammonia  alum 50  g. 

Water 400  c.c. 

Glycerin 100  c.c. 

Methyl  alcohol 100  c.c. 

Alum-hematoxylin — 

1 .  Hematoxylin 2  g. 

Absolute  alcohol 100  c.c. 

2.  Ammonia  alum 2  g. 

Water 100  c.c. 

Mix  i  and  2  and  add — 

Glycerin 850  c.c. 

Glacial  acetic  acid 100  c.c. 

Allow  to  stand  for  one  month  before  using. 
Bismarck  brown — 

Bismarck  brown o .  5  g. 

Water 100  c.c. 

Sajranin — 

Safranin 0.5  g. 

Water 100  c.c. 

Carbolic  thionin-blue  (Nicolle') — 

Thionin-blue i  g. 

Carbolic  acid 2 . 5  g. 

Water 100  c.c. 

Alum  carmin — 

Alum 2 . 5  g. 

Carmin i  •  o  g. 

Water 100  c.c. 

132 


STAINING  SOLUTIONS  133 

Lithium-carmin  (Orth) — 

Carmin 2 . 5  g. 

Saturated  watery  solution  of  lithium  car- 
bonate   100  c.c. 

Add  a  few  crystals  of  thymol. 
Kiihne's  methylene-blue — 

Methylene-blue i .  5  g. 

Absolute  alcohol 10  c.c. 

Carbolic  acid  solution  (5$) 100  c.c. 

Carbolic  gentian-violet  (Nicolle) 

Gentian-violet  (sat.  alcoh.  sol.) 10  c.c. 

Carbolic  acid i  g. 

Water po  c.c. 


APPENDIX  III 

FROST'S  CULTURE  CHART  (MODIFIED)' 

Group 

Name  of  organism 

Source,  habitat,  etc 


MORPHOLOGICAL  CHARACTERS: 

i 

Incubation 
Temp.  (°C.) 

Sketches 

i  .    Form  : 
a     Broth 

b     Agar 

c     Gelatin 

d     Other  Media         

2.    Size     

3.    Cell  groupings  and  arrangements  in 
growths 

4     Staining  powers 

d     ^nilin-gentian-violet  .. 

b     Loeffler's  methylene-blue 

c.    Gram's  stain  

d.    Special  stains  
5.    Motility  

a     Character  of  movement 

b     Flagella  stain 

6     Spores 

7.    Special  characters,  such  as       

deposits,  vacuoles  
pleomorphic  and  involution  forms, 
capsules  etc 

PHYSIOLOGICAL  CHARACTERS 
i.    Relation  to  temperature 


Relation  to  free  oxygen. 

Relation  to  other  agents,  such  as  dessica- 

tion,  light,  disinfectants,  etc 


1  This  represents  a  model  only.     Open  spaces  and  lines  should  be  extended  for 
practical  use  to  allow  sufficient  room  for  descriptions. 

134 


FROST'S  CULTURE  CHART 


4.  Pigment  production 

5.  Growth  in  carbohydrate  media: 
a.    Stab  or  shake  culture 

6.    Fermentation-tube:  i.  Growth  in  bulb.  .  .  .2.  In  closed  arm.  . 

c.  Percentage  gas  produced  in:   Dextrose. ..  .Lactose .  .  .Saccharose 

After  24  hours. . 

After  48  hours 

TT 

Gas  formula  T^T~ 

CU2 

d.  Reaction  in  bulb 2.  Closed  arm 

6.  Acid  or  alkali  production,  in  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites to  ammonia 

8.  Indol  production;  24  hours 48  hours 4  days 

fecal  odor;  24  hours 48  hours 4  days 

9.  Enzym  production;   coagulative proteolytic diastatic 

10.  Characteristic  odor 

11.  Pathogenesis 


CULTURAL  CHARACTERS  OF: 


(I) 

Gelatin 
plate  or 
roll  tube 

a)  Surface 
colonies 

b)  Deep 
colonies 


(2) 

Agar  plate, 

or  roll 

tube 
a)    Surface 

colonies 
6)    Deep 

colonies 


Reaction  of 

Medium 
Incubation 
Temp.  (°C) 


Description 


Sketches 


136    LABORATORY   GUIDE   IN   BACTERIOLOGY 
CULTURAL  CHARACTERS  OF: 


Reaction, 
etc. 

24  Hrs. 

48  Hrs. 

6  Days 

Sketches 

(3) 

Agar  streak 
and  stab 

A 

/  \ 
/      \ 

A 

/        X 

/      \ 

/          \ 

(4) 
Potato 

& 

^x             / 

(5) 

Broth 

i 



v| 

- 

(6) 

i 

Litmus  milk 

L 

w 

(7) 

Gelatin  stab 

^ 

FROST'S   CULTURE   CHART 
CULTURAL  CHARACTERS  OF: 


Reaction, 
etc. 

24  Hrs. 

48  Hrs. 

6  Days 

Sketches 

(8) 

Glucose-agar 
or  special 

media 

INDEX 


Absorbent  cotton  filter  17 

Acid,  chromic  8 

Acid-resisting  bacilli  103 
Actinomyces  asteroides,  bovis,  etc.  106 

Agar-agar  1 1 

Agar,  beerwort  128 

Agar,  bile-salt  128 

Agar,  blood  128 

Agar,  glucose  19 

Agar,  glycerin  128 

Agar,  litmus-lactose  127 

Agar,  litmus-mannit  127 

Agar,  neutral  red  127 

Agar,  preparation  of  n 

Agar-slants  39 

Agglutination  87 
Air, bacterial  examination  of  37, 114,118 

Air,  bacteria  from  37 

Alkaline  blood-serum  128 

Alum  carmin  132 

Alum  hematoxylin  132 

Amylase  65 

Anaerobic  cultivation  107 

Anaerobic  group  107 

Analysis  of  gas  82 

Anilin-gentian-violet  30 

Anthrax  bacillus  98 

Anthrax  group  98 

Arnold  steam  sterilizer  24 

Aspirator  46 

Autoclav  20 

Autopsy  75 

Bacilli,  acid-resisting  103 

Bacillus  acidi  lactici  79 

Bacillus  aerogenes  capsulatus  107 

Bacillus  anthracis  98 
Bacillus  anthracis  symptomatici  107 

Bacillus  botulinus  107 
Bacillus  cholerae  suis  79,84 
Bacillus  cloacae  79,  89 

Bacillus  coli  79 

Bacillus  cuniculicida  97 

Bacillus  der  Rinderseuche  97 

Bacillus  der  Schweineseuche  97 

Bacillus  diphtheriae  94 
Bacillus  dysenteriae  79,  85 
Bacillus  enteritidis  79,  84 
Bacillus  faecalis  alcaligenes  79,  85 

Bacillus  Gaertner's  84 

Bacillus,  grass  103 

Bacillus  icteroides  79 
Bacillus  lactis  aerogenes  79,  92 

Bacillus  leprae  103 

Bacillus  mallei  105 


PAGE 

Bacillus  mucosus  capsulatus  92 

Bacillus  oedematis  maligni  107 

Bacillus  of  bubonic  plague  97 

Bacillus  of  dysentery  79,  85 

Bacillus  of  rabbit  septicemia  97 

Bacillus  paratyphosus  79,  84 

Bacillus  pestis  97 

Bacillus  prodigiosus  69 

Bacillus  pseudo-diphthericus  94 

Bacillus  pyocyaneus  69 

Bacillus  smegrnae  103 

Bacillus  subtilis  98 

Bacillus  tetani  107 

Bacillus  tuberculosis  103 

Bacillus  typhosus  79,85 

Bacillus  violaceus  69 

Bacillus  xerosis  94 

Bacteria,  chromogenic  69 

Bacteria  from  air  37,  118 

Bacteria,  pyogenic  73,  76 

Bacteria,  unknown  112 
Bacterial  examination  of  air 

37,  114,  118 

Bacterial  examination,  of  milk  114,  119 
Bacterial  examination,  of  water  114 
Bacterium  termo  79 
Balsam  bottle  4 
Balsam,  Canada  44 
Basket,  wire  4 
Beerwort-agar  128 
Beerwort-gelatin  128 
Berkefeld  filter  46 
Bile-salt  agar  128 
Bile-salt  broth  129 
Bismarck  brown  132 
Blood-agar  128 
Blood-serum  125 
Blood-serum  alkaline  128 
Blood-serum,  dog's  131 
Bouillon  (see  peptone  broth)  25 
Bread-paste  medium  128 
Broth,  bile-salt  129 
Broth,  glycerin  127 
Broth,  litmus  128 
Broth,  meat  26 
Broth,  nitrate  131 
Brothr  peptone  25 
Broth,  preparation  of  25 
Broth,  sugar- free  27,  79 
Brownian  movement  43 
Bubonic  plague  97 
Buchner's  anaerobic  culture  meth- 
od 107 
Butter,  bacilli  in  103 


139 


1 4o 


INDEX 


Canada  balsam  44 

Capaldi's  egg-medium  130 

Capsulated  group  92 

Capsule  stain  76 

Capsule  stain,  Friedlander's  77 

Capsule  stain,  Welch's  77 
Capsule  stain,  Welch's  modified     no 

Carbol-fuchsin  30 

Carbolic  gentian- violet  133 

Carbolic  thionin-blue  132 

Carmin  43 

Carmin,  alum  132 

Carmin,  lithium  133 

Casein  64 

Caseinogen  64 

Cedar  oil  35 
Cholera  red  reaction  (see  Indol)      101 

Chrompgenic  group  69 

Clarifying  media  13 
Cleaning  glassware 

Cleaning  mixtures  8 
Coagulation  64 
Coagulative  enzym  64 
Colon  bacillus  79 
Colon  group  79 
Colonies,  counting  of  116 
Colony  39 
Condensation  water  40 
Conradi's  medium  130 
Cotton  filter  47 
Cotton  filter,  absorbent  17 
Counting  colonies  116 
Culture- charts  description  57 
Culture  media  n 
Culture- tubes  3,  4,  14 
Culture-tubes,  plugging  of 
Culture-tubes,  potato  3,  28 
Cultures,  egg  95 
Cultures,  plate  70 
Cultures,  pure  39 
Death-point,  thermal  124 
Decolorization  of  litmus  65 
Delafield's  hematoxylin  132 
Description  of  colonies  57 
Description  of  cultures  57 
Dextrose  (see  under  glucose) 
Dextrose  yeast  water  130 
Diagnosis  of  glanders  (Strauss)  105 
Diastase  65 
Diastatic  enzym  65 
Dilution  tables  117 
Diphtheria  bacillus  94 
Diphtheria  group  94 
Diphtheria  toxin  96 
Directions  for  filling  out  culture- 
charts  6 1 
Directions,  general  2 
Discontinuous  sterilization  24 
Disinfectants  122 
Dog's  blood-serum  131 
Dorset's  egg  medium  130 
Dorset's  egg-yolk  medium  130 


PAGE 

Drigalski's  medium  130 

Dung  bacillus  103 

Dunham's  peptone  solution  25 
Dysentery  bacillus  79,  85 

Egg  cultures  95 

Egg  media  130 

Ehrlich's  anilin -gentian- violet  30 

Eisner's  medium  129 

Endo's  medium  130 
Enriching  method  of  Schottelius  101 
Enriching  solution  (Hoffmann  and 

Fischer)  130 

Enzym  64 

Enzym,  coagulative  64 

Enzym,  diastatic  65 

Enzym,  production  of  64 

Enzym,  rennet-like  64 

Erlenmeyer  flask  5 
Examination,  bacterial  of  air 

37,  114,118 

Examination,  bacterial  of  milk  1 14, 1 19 
Examination,  bacterial  of  water  114 
Fermentation-tube  4 
Filament  formation  99 
Filling  culture-tubes  14 
Filter,  absorbent  cotton  17 
Filter,  Berkefeld  46 
Filter,  cotton  47 
Filter,  paper  15 
Filter  rack  17 
Filtering  media  16 
Filtering  media  by  vacuum  17 
Finkler  and  Prior's  spirillum  101 
Flagella,  staining  of  86 
Flask,  Erlenmeyer  5 
Fowl  cholera,  bacillus  of  97 
Friedlander's  capsule  stain  77 
Friedlander's  pneumobacillus  92 
Frost's  fermentation-chart  81 
Frost's  culture-chart  (modified)  134 
Fruit-jar  method  of  anaerobic  cul- 
tivation 1 08 
Fuchsin  30 
Fuller's  standard  13 
Gaertner's  bacillus  84 
Garden  earth,  inoculation  of  1 1 1 
Gas  analysis  82 
Gas  formula  82 
Gas  generator  109 
Gasometer  (Frost's)  81 
Gelatin,  beerwort  128 
Gelatin,  glucose  128 
Gelatin,  liquefaction  of  64 
Gelatin,  litmus-lactose  127 
Gelatin,  peptone  23 
Gelatin,  whey  128 
General  directions  2 
Gentian- violet  30 
Gentian-violet,  carbolic  133 
Germination  of  spores  51 
Glanders  105 
Glassware,  cleaning 


INDEX 


141 


PAGE 

Glassware,  sterilization  9 

Glucose-agar  19 

Glucose-gelatin  127 

Glycerin-agar  128 

Glycerin-broth  127 

Glycerinated  potato  129 

Glycogen  27 

Gonorrheal  pus  76 

Gram's  iodin  solution  30 

Gram's  stain  53 

Grass  bacillus  103 

Green  mold  51 
Gruber-Widal  test  for  typhoid  fever  87 

Gypsum  blocks  5° 

Hand -lens  5 

Hanging-drop  42 

Hay-infusion  129 

Hematoxylin  alum  132 

Hematoxylin,  Delafield's  132 

Hemorrhagic  septicemia  group         97 

Hill's  test -rods  122 
Hoffmann  and  Fischer's  enriching 


method 
Hog-cholera  group 
Hot-air  sterilizer 
Hydrogen  gas-generator 
Immersion  oil 
Impression  preparation 
Incubator 
Indol 
Indol  test 

Infection,  phenomena  of 
Influence  of  disinfectants 
Influence  of  moist  heat 
Influence  of  sunlight 
Infusion,  hay 


130 

79,84 

9 

109 


I! 

85 

45 

122 
124 
123 
129 

Inoculation  (see  various  headings) 

79,  84 
79,  84 
24 
79 
102 
92 
73 


Intermediate  group 
Intermittent  sterilisation 
Intestinal  group 
Intramuscular  inoculation 
Intraperitoneal  inoculation 
Intravenous  inoculation 
Involution  forms 
Iodin  solution,  Gram's 
Isolation  of  unknown  bacteria 
Jar,  Novy 
Klatschprdparat 
Kiihne's  methylene-blue 
Labeling  media 
Laboratory  rules 
Lactose-litmus-agar 
Lactose-litmus-gelatin 
Liquefaction  of  casein 
Liquefaction  of  gelatin 
Lithium  carmin 
Litmus-broth 
Litmus,  decolorization  of 
Litmus-lactose-agar 
Litmus-lactose-gelatin 
Litmus-mannit-agar 


30 

112 
1 08 

98 
133 

20 
I 

127 
127 

64 

64 
133 
128 

65 
127 
127 
127 


PAGE 

Litmus  milk  28 

Litmus-solution  127 

Litmus- whey  128 

Loeffler's  flagella  stain  86 

Loeffler's  methylene-blue  30 

Mac  Conkey's  bile-salt  agar  128 

Mac  Conkey's  bile-salt  broth  129 

Magnifier  5 

Mannit  litmus  agar  127 

Meat  broth  26 

Meat-press  26 

Media,  adjusting  reaction  of  14 

Media,  adjusting  weight  of  13 

Media,  clarifying  13 

Media,  filling  in  tubes  14 

Media,  filtering  14 

Media,  filtering  by  vacuum  18 

Media,  phenol  131 

Media,  preparation  of  1 1 

Media,  preservation  of  28 

Media,  reaction  of  12 

Media,  special  125 

Media,  standard  methods  125 

Medium,  bread-paste  128 

Medium,  Drigalski-Conradi's  130 

Medium,  Eisner's  129 

Medium,  Endo's  130 

Medium,  Uschinsky's  131 

Medium,  wine-must  129 

Method  of  describing  cultures  39 
Method  of  inoculation  of  media        57 

Methylene-blue,  Kiihne  133 

Methylene-blue,  Loeffler  30 

Micrococcus  gonorrhoeae  76 
Micrococcus  intracellularis  menin- 

gitidis  76 

Micrococcus  lanceolatus  76 

Micrococcus  melitensis  105 

Micrococcus  tetragenus  73 

Micrococcus  zymogenes  76 

Microscope,  description  of  31 

Milk,  acid-resisting  bacilli  in  104 
Milk,  bacterial,  examination  of  114,119 

Milk,  litmus  28 

Milk,  pasteurization  of  119 

Milk,  sterilization  of  119 

Moeller's  grass  bacillus  103 

Moeller's  spore  stain  98 

Moist  heat,  influence  of  124 
Moist  heat,  sterilization  by         20,  24 

Mold,  green  51 

Mold  spores  39 

Molds  49 

Molds,  staining  of  5° 

Molecular  movement  43 
Mordant 

Mouse,  inoculation  of 

Mouse- holder  78 

Muscle-sugar  27 

Needles,  platinum  4 

Neisser's  stain  94 

Neutral  red  agar  127 


I42 


INDEX 


PAGE 

Nicole's  carbolic  gentian-violet       133 
Nicole's  carbolic  thionin-blue         132 

Nitrate  broth  131 

Nitrate  solution  131 

Nitrites  63 

Nitrites,  test  for  83 

Nitroso-indol  reaction  101 

Normal  solution  13 

Novy  jar  108 

Oil,  immersion  34 

Orth's  lithium  carmin  133 

Parietti's  solution  129 
Park's  anaerobic  culture  method     107 

Pasteurization  of  milk  119 

Pedesis  43 

Penicillium  glaucum  5 1 

Peptone  broth  25 

Peptone  gelatin  23 

Peptone  solution,  Dunham's  25 

Peptonization  64 

Petri  dishes  5 

Phenol  media  131 

Phenolphthalein  12 

Phenomena  of  infection  45 

Phenomena  of  sterilization  45 

Pigments  7 1 

Plate  cultures  70 

Platinum  needles  4 

Plugging  culture-tubes  8 

Pneumobacillus  92 

Pocket  inoculation  96 
Polar  staining                              85,  97 

Potato,  glycerinated  129 

Potato,  preparation  of  27 

Potato-tubes  28 

Preparation,  Gram  53 

Preparation,  impression  98 

Preparation  of  agar-agar  1 1 

Preparation  of  bouillon  25 

Preparation  of  broth  25 

Preparation  of  culture  media  1 1 
Preparation  of  Dunham's  peptone 

solution  25 

Preparation  of  gelatin  23 

Preparation  of  glucose- agar  19 
Preparation  of  stains  (see  various 

headings) 

Preparation  of  peptone  solution        25 

Preparation  of  potato  27 

Preparation,  stained  44 

Preservation  of  media  1 1 

Proteolysis  64 

Proteus  group  89 

Pure  culture  39 

Pus,  gonorrheal  76 

Pyocyanin  72 

Pyogenic  group  A  73 

Pyogenic  group  B  76 

Rabbit  septicemia  97 

Reaction,  indol  83 

Reaction,  nitrites  83 

Reaction,  nitroso-indol  101 


Reaction  of  media  14 

Rennet-like  enzym  64 

Rinderpest  97 

Routine  study  53 

Saccharomyces  cerevisiae  49 

Safranin  132 

Sarcina  lutea  69 

Schottelius'  enriching  method  101 

Schweineseuche  97 

Soap-powder  8 

Solution,  Dunham's  peptone  25 

Solution,  litmus  127 

Solution,  nitrate  131 

Solution,  normal  13 

Solution,  Parietti's  129 
Solutions,  staining  30,  132 

Special  media  125 

Spirillum  cholerae  asiaticae  101 

Spirillum  Metchnikovi  101 
Spirillum  of  Finkler  and  Prior  101 

Spirillum  tyrogenum  101 

Spore-staining  08 

Spores,  germination  of  51 

Spores  of  bacteria  20 

Spores  of  yeast  51 

Spores  of  molds  39 

Stain,  Friedlander's  capsule  77 

Stain,  Welch's  capsule  77 
Stain,  Welch's  capsule  modified  no 

Stain,  Gram's  53 

Stain,  Loeffler's  flagella  86 

Stain,  Neisser's  94 

Stained  preparation  44 

Staining  acid-resisting  bacilli  103 

Staining  flagella  86 

Staining  molds  51 
Staining,  polar  85,  97 
Staining  solutions  30,  132 

Staining  spores  98 

Standard,  Fuller's  13 
Standard  method  of  preparing 

media  125 

Staphylococcus  pyogenes  albus  73 
Staphylococcus  pyogenes  aureus  73 

Starch  65 

Steam  sterilizer  24 

Sterilization  9 

Sterilization,  discontinuous  24 

Sterilization,  intermittent  24 

Sterilization  of  glassware  9 

Sterilization  of  milk  1 19 

Sterilization,  phenomena  of  45 

Sterilizer,  hot-air  9 

Sterilizer,  Arnold  steam  24 
Strauss  method  of  diagnosis  of 

glanders  105 

Streptococcus  pyogenes  73 

Study,  routine  53 

Subcutaneous  inoculation  85 
Sugar-free  broth  27,  79 

Sulphur  granules  106 

Sunlight,  influence  of  123 


INDEX 


PAGE 

Tables,  dilution 

17 

Tetanus  toxin 

no 

Test  for  indol 

83 

Test  for  nitrites 

83 

Test-rods,  Hill's 

122 

Thermal  death-point 

124 

Thermostat 

55 

Thionin-blue 

132 

Torulae 

49 

Tube,  culture 

4 

Tube,  fermentation 

4 

Tube,  potato  culture 

28 

Tubes,  plugging  of 
Typhoid-dysentery  group            79 
Uschinsky's  medium 

8 
,  85 
I31 

Water,  bacterial  examination  of 

114 

PAGE 

40 
77 
no 
64 
128 
128 


Water  of  condensation 
Welch's  capsule  stain 
Welch's  capsule  stain  modified 
Whey 

Whey-gelatin 
Whey  litmus 

Widal  test  (see  Gruber-Widal  test) 
Wine-must  129 

Winogradsky's  solution  129 

Xylol  33,  35 

Yeast  water  130 

Yeast  water,  dextrose  130 

Yeasts  39,  49,  51 

Yeasts,  budding  of  51 

Yeasts,  spores  of  51 

Ziehl-Neelsen's  carbol-fuchsin  30 


YB 


LIBRARY 
G 


144184 


H<4 


THE  UNIVERSITY  OF  CALIFORNIA  LIBRARY 


